Full Ebook of Circadian Regulation Methods and Protocols Methods in Molecular Biology 2482 Guiomar Solanas Online PDF All Chapter
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Methods in
Molecular Biology 2482
Circadian
Regulation
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire Hatfield,
Hertfordshire, UK
Edited by
Guiomar Solanas
Institute for Research in Biomedicine, Barcelona Institute for Science and Tech, Barcelona, Spain
Patrick-Simon Welz
Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain
Editors
Guiomar Solanas Patrick-Simon Welz
Institute for Research in Biomedicine Hospital del Mar Medical Research Institute (IMIM)
Barcelona Institute for Science and Tech Barcelona, Spain
Barcelona, Spain
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
This book compiles a series of methods on several aspects of circadian research. These
methods are thoroughly explained for newcomers to the field and contain some of the latest
techniques for expert scientists to be updated. The reader will find protocols covering the
wide variety of daily rhythmic processes, using diverse model organisms, for the analysis of
circadian rhythms in the SCN and in peripheral organs, describing in vitro systems and in
silico methods.
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
vii
viii Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Contributors
JOSE M. ADROVER • Area of Cell and Developmental Biology, Fundacion Centro Nacional de
Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, USA
FOREST AGOSTINELLI • Institute for Genomics and Bioinformatics, University of California,
Irvine, CA, USA; Department of Computer Science and Engineering, University of South
Carolina, Columbia, SC, USA
ALEJANDRA AROCA-CREVILLÉN • Area of Cell and Developmental Biology, Fundacion Centro
Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
ANNA ARTATI • Metabolomics and Proteomics Core Facility, Helmholtz Zentrum München,
German Research Center for Environmental Health (GmbH), Neuherberg, Germany
DANIELA BACHETTI • Department of Biomedical Sciences, University of Windsor, Windsor,
ON, Canada
PIERRE BALDI • Department of Computer Science, University of California Irvine, Irvine,
CA, USA; Institute for Genomics and Bioinformatics, University of California, Irvine,
CA, USA
ANDREW D. BEALE • MRC Laboratory of Molecular Biology, Cambridge, UK
ERIC L. BITTMAN • Department of Biology and Program in Neuroscience & Behavior,
University of Massachusetts, Amherst, MA, USA
SUSANNA CAMPESAN • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
YUHUI CAO • Department of Pharmacology and Systems Physiology, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
VANIA CARMONA-ALCOCER • Department of Biomedical Sciences, University of Windsor,
Windsor, ON, Canada
LEANDRO CASIRAGHI • Department of Biology, University of Washington, Seattle, WA, USA
AMANDINE CHAIX • Department of Nutrition and Integrative Physiology, University of Utah,
Salt Lake City, UT, USA
SUNG KOOK CHUN • Department of Biological Chemistry, Center for Epigenetics and
Metabolism, Chao Family Comprehensive Cancer Center, University of California, Irvine
(UCI), Irvine, CA, USA
LAURA DELFINO • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
KENNETH ALLEN DYAR • German Center for Diabetes Research (DZD), Neuherberg,
Germany; Metabolic Physiology, Institute for Diabetes and Cancer (IDC), Helmholtz
Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany
GIORGIO FEDELE • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
ANNA-MARIE FINGER • Charité Universit€ a tsmedizin Berlin, Berlin, Germany; Freie
Universit€ at Berlin, Berlin, Germany; Humboldt-Universit€ a t zu Berlin, Berlin, Germany;
Laboratory of Chronobiology, Berlin Institute of Health, Berlin, Germany
DAVID GATFIELD • Center for Integrative Genomics, University of Lausanne, Lausanne,
Switzerland
xi
xii Contributors
DOMINIK LUTTER • Computational Discovery Research, Institute for Diabetes and Obesity
(IDO), Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany;
German Center for Diabetes Research (DZD), Neuherberg, Germany
PALOMA MAS • Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-
UB, Barcelona, Spain; Consejo Superior de Investigaciones Cientı́ficas (CSIC), Barcelona,
Spain
SELMA MASRI • Department of Biological Chemistry, Center for Epigenetics and Metabolism,
Chao Family Comprehensive Cancer Center, University of California, Irvine (UCI),
Irvine, CA, USA
JENNIFER W. MITCHELL • Department of Cell and Developmental Biology and Neuroscience
Program, Beckman Institute for Advanced Science and Technology, University of Illinois at
Urbana-Champaign, Urbana, IL, USA
KAZUTOSHI MIYASHITA • Department of Endocrinology, Metabolism, and Nephrology, Keio
University School of Medicine, Tokyo, Japan
GREGOR MÖNKE • European Molecular Biology Laboratory, Heidelberg, Germany
JOHN S. O’NEILL • MRC Laboratory of Molecular Biology, Cambridge, UK
MASAAKI OKADA • Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-
UAB-UB, Barcelona, Spain
IWONA OLEJNICZAK • Institute of Neurobiology, Center of Brain, Behavior & Metabolism,
University of Lübeck, Lübeck, Germany
MARTA DEL OLMO • Institute for Theoretical Biology, Charite and Humboldt Universit€ at zu
Berlin, Berlin, Germany
ALEJANDRO OSORIO-FORERO • Department of Fundamental Neurosciences, University of
Lausanne, Lausanne, Switzerland
HENRIK OSTER • Institute of Neurobiology, Center of Brain, Behavior & Metabolism,
University of Lübeck, Lübeck, Germany
KATHYANI PARASRAM • Department of Biomedical Sciences, University of Windsor, Windsor,
ON, Canada
MIRI PARK • Department of Pharmacology and Systems Physiology, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
PAUL PETRUS • Center for Epigenetics and Metabolism, U1233 INSERM, Department of
Biological Chemistry, University of California, Irvine, CA, USA
VIOLETTA PILORZ • Institute of Neurobiology, Center of Brain, Behavior & Metabolism,
University of Lübeck, Lübeck, Germany
CORNELIA PREHN • Metabolomics and Proteomics Core Facility, Helmholtz Zentrum
München, German Research Center for Environmental Health (GmbH), Neuherberg,
Germany
EZIO ROSATO • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
MUNTAHA SAMAD • Department of Computer Science, University of California Irvine,
Irvine, CA, USA; Institute for Genomics and Bioinformatics, University of California,
Irvine, CA, USA
UELI SCHIBLER • Department of Molecular Biology, Faculty of Sciences, University of Geneva,
Geneva, Switzerland
CHRISTOPH SCHMAL • Institute for Theoretical Biology, Humboldt Universit€ a t zu Berlin,
Berlin, Germany
FLORE SINTUREL • Division of Endocrinology, Diabetes, Nutrition and Patient Education,
Department of Medicine, University Hospital of Geneva, Geneva, Switzerland;
xiv Contributors
Abstract
Human sleep is regulated by light in two fundamental ways: The light-dark (LD) cycle entrains a circadian
clock that in turn regulates sleep timing, and light per se can acutely inhibit sleep. Throughout evolution,
these sleep regulatory systems became highly sensitive to the effects of light and they can be affected by the
relatively low light intensities that are used indoors. Thus, postindustrial living conditions have created built
environments that have isolated humans from the natural LD cycle and exposed them to an artificial one
that can affect daily sleep timing. Studying indigenous communities that have differential access to
electricity, as well as communities living in highly urbanized areas, we and others have shown that human
access to artificial light has delayed the daily onset of sleep but has had a smaller effect on its offset, leading
to an overall reduction in sleep duration that is pervasive in modern societies. In this chapter we discuss
these studies, highlight their main findings, and point to their limitations.
Key words Sleep, Artificial light, Indigenous communities, Preindustrial societies, Modern societies
Guiomar Solanas and Patrick-Simon Welz (eds.), Circadian Regulation: Methods and Protocols,
Methods in Molecular Biology, vol. 2482, https://doi.org/10.1007/978-1-0716-2249-0_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
1
2 Leandro Casiraghi and Horacio O. de la Iglesia
Fig. 1 The access to electric light delays sleep timing. Clock plots displaying light exposure and actimetry-
based sleep timing. The plot on the left is from participants in a Toba/Qom community that has no access to
electricity. The plot on the right is from participants living in a highly urbanized community (Seattle, WA, USA)
and sampled during the weekend. Green bars represent the interquartile range for the first and last times that
individuals are exposed to a light intensity of 50 lux. Purple and blue bars represent the times of sleep onset
and offset, respectively. Note that in both cases sleep starts after the last exposure to 50 lux, but this occurs
much later relative to solar time in the highly urbanized community, in which the sleep onset occurs after solar
midnight. Gray and white in the pie represent the natural night and day, respectively. Time represents the local
clock time
Sleep Under Preindustrial Conditions: What We Can Learn from It 3
Summary of studies classified by the population studied and the research team or teams. When more than one work is referenced for the same
population and team, methods and main findings are summarized indistinctly for the sake of simplicity. Rows are ordered chronologically
Population/
community Region Type of study Methodology Main findings References
Trobriand Tauwema Observational, population with Actimetry Young infants displayed ultradian sleep [21]
islanders Village, Papúa no access to electricity only patterns and slept more. Wives in couples
Nueva Guinea slept more than their husbands.
Mapuche Neuquén, Observational, population with Sleep diaries Wake-up times are delayed in the winter. [18]
Argentina no access to electricity only
Rural village Paraná State, Controlled, access to electric Questionnaires, Adolescents with no access to electric light [19, 33, 34]
adolescents Brazil light vs. no access; different actimetry, slept more and showed no sleep deprivation.
school times DLMO School times correlated with sleep duration
and onset. Access to electric light correlated
with delayed phase of sleep.
Egyptian villagers El Cairo, Egypt Observational Diaries Cosleeping correlated with less sleep but of [17, 35]
better quality. High occurrence of daytime
Leandro Casiraghi and Horacio O. de la Iglesia
napping.
Haitian rural Fondwa, Haiti Observational, population with Actimetry Sleep fragmentation decreased with age. [36]
villagers no access to electricity only Longer times of staying-in-bed than in
industrialized societies, but similar effective
sleep duration.
Toba/Qom Formosa, Controlled, different levels of Actimetry, diaries, Access to electricity correlated with delayed [22, 24, 25]
Argentina access to electric light and DLMO sleep onset and reduced sleep duration.
urbanity Larger differences between electricity levels in
the winter.
Electric light associated with delayed onset of
melatonin release during the winter.
Amazonian Amazonian Controlled, access to electric Diaries, actimetry, Access to electricity and urbanization [26, 37, 38]
rubber tappers Extractive light vs. no access DLMO correlated with delayed and shorter sleep.
Reserve, Brazil
San people Northeastern Mixeda, communities with no Actimetry, Shorter and later sleep in the summer. Low [20]
Namibia access to electric light interviews incidence of insomnia.
Tsimane people Beni Mixeda, communities with no Actimetry, Shorter and later sleep in the summer. Low [20]
Department, access to electric light interviews incidence of insomnia.
Bolivia
Hadza people Northern Observational, mixeda Actimetry, Short sleep duration compared to industrial [20, 23, 39]
Tanzania interviews societies. Communal “sentinel” pattern of
sleep.
Rural villagers Baependi, Brazil Observational Actimetry, DLMO, One of the earliest chronotypes in literature. [40, 41]
questionnaires Women slept more than men.
Malagasy rural Mandena, Observational, population with Actimetry, PSG High prevalence of “segmented” night sleep [42]
villagers Madagascar no access to electric light and daytime napping. Short sleep duration
only compared to industrial societies.
Milange and Zambézia, Controlled, urban vs. rural Actimetry Urbanity was associated with delayed sleep [28]
Tengua Mozambique conditions onset. No differences in sleep duration.
communities
Quilombolas Southern Brazil Controlled, different levels of Actimetry, MCT Access to electricity and urbanization [27]
access to electric light and Questionnaire correlated with delayed and shorter sleep.
urbanity
Indigenous Tanna Island, Controlled, access to electric Actimetry Access to electricity associated with sleep onset [29]
Melanesians Vanuatu light vs. no access and reduces sleep duration and efficiency.
a
Yetish et al. [20] compares these three preindustrial populations
Sleep Under Preindustrial Conditions: What We Can Learn from It
9
10 Leandro Casiraghi and Horacio O. de la Iglesia
6 Conclusions
The last two decades have seen a marked increase in studies that
have capitalized on the access to communities living in a range of
preindustrial conditions—from hunting-gathering to agricultural
peoples—to gain insight into sleep patterns in the absence of
urbanization. These studies have provided unequivocal evidence
that urbanization, and its associated increased exposure to electric
light, delays daily sleep timing and, in many cases, shortens its
12 Leandro Casiraghi and Horacio O. de la Iglesia
Acknowledgments
We are thankful to Isabelle Hua and Gideon Dunster for their help
with Fig. 1, and to Dr. Ray Sanchez for comments on the original
manuscript. Supported by NSF RAPID award #1743364 and Lea-
key Foundation grant #1266 to HOD.
References
1. Tillman DA (2018) Coal-fired electricity and resetting of the human circadian pacemaker.
emissions control: efficiency and effectiveness. Science 244(4910):1328–1333
Elsevier Science & Technology, Saint Louis 10. Klerman EB, Gershengorn HB, Duffy JF, Kro-
2. Boivin DB, Duffy JF, Kronauer RE, Czeisler nauer RE (2002) Comparisons of the variabil-
CA (1996) Dose-response relationships for ity of three markers of the human circadian
resetting of human circadian clock by light. pacemaker. J Biol Rhythm 17(2):181–193
Nature 379(6565):540–542. https://doi. 11. Wright KP Jr, McHill AW, Birks BR, Griffin
org/10.1038/379540a0 BR, Rusterholz T, Chinoy ED (2013) Entrain-
3. Hughes S, Jagannath A, Hankins MW, Foster ment of the human circadian clock to the natu-
RG, Peirson SN (2015) Photic regulation of ral light-dark cycle. Curr Biol 23(16):
clock systems. Methods Enzymol 552:125– 1554–1558. https://doi.org/10.1016/j.cub.
143. https://doi.org/10.1016/bs.mie.2014. 2013.06.039
10.018 12. Stothard ER, McHill AW, Depner CM, Birks
4. Johnson CH, Elliott JA, Foster R (2003) BR, Moehlman TM, Ritchie HK, Guzzetti JR,
Entrainment of circadian programs. Chrono- Chinoy ED, LeBourgeois MK, Axelsson J,
biol Int 20(5):741–774 Wright KP Jr (2017) Circadian entrainment
5. Chang AM, Aeschbach D, Duffy JF, Czeisler to the natural light-dark cycle across seasons
CA (2015) Evening use of light-emitting eRea- and the weekend. Curr Biol 27(4):508–513.
ders negatively affects sleep, circadian timing, https://doi.org/10.1016/j.cub.2016.12.041
and next-morning alertness. Proc Natl Acad Sci 13. Dunster GP, de la Iglesia L, Ben-Hamo M,
U S A 112(4):1232–1237. https://doi.org/ Nave C, Fleischer JG, Panda S, de la Iglesia
10.1073/pnas.1418490112 HO (2018) Sleepmore in Seattle: later school
6. Kleitman N (1939) Sleep and wakefulness as start times are associated with more sleep and
alternating phases in the cycle of existence. The better performance in high school students. Sci
University of Chicago Press, Chicago Adv 4(12):eaau6200. https://doi.org/10.
7. Aschoff J (1965) Circadian rhythms in man. 1126/sciadv.aau6200
Science 148:1427–1432 14. van de Wouw E, Evenhuis HM, Echteld MA
8. Aschoff J, Fatranska M, Giedke H, Doerr P, (2013) Comparison of two types of Actiwatch
Stamm D, Wisser H (1971) Human circadian with polysomnography in older adults with
rhythms in continuous darkness: entrainment intellectual disability: a pilot study. J Intellect
by social cues. Science 171(967):213–215 Develop Disabil 38(3):265–273. https://doi.
org/10.3109/13668250.2013.816274
9. Czeisler CA, Kronauer RE, Allan JS, Duffy JF,
Jewett ME, Brown EN, Ronda JM (1989) 15. Rupp TL, Balkin TJ (2011) Comparison of
Bright light induction of strong (type 0) Motionlogger Watch and Actiwatch actigraphs
Sleep Under Preindustrial Conditions: What We Can Learn from It 13
34. Pereira ÉF, Louzada FM, Moreno CR (2010) 39. Crittenden AN, Samson DR, Herlosky KN,
Not all adolescents are sleep deprived: a study Mabulla IA, Mabulla AZP, McKenna JJ
of rural populations. Sleep Biol Rhythms 8(4): (2018) Infant co-sleeping patterns and mater-
267–273. https://doi.org/10.1111/j. nal sleep quality among Hadza hunter-
1479-8425.2010.00458.x gatherers. Sleep Health 4(6):527–534.
35. Worthman CM, Brown RA (2012) Sleep bud- https://doi.org/10.1016/j.sleh.2018.10.005
gets in a globalizing world: biocultural interac- 40. Beijamini F, Knutson KL, Lorenzi-Filho G,
tions influence sleep sufficiency among Egan KJ, Taporoski TP, De Paula LK, Negrao
Egyptian families. Soc Sci Med. https://doi. AB, Horimoto AR, Duarte NE, Vallada H,
org/10.1016/j.socscimed.2012.03.048 Krieger JE, Pedrazzoli M, Pereira AC, von
36. Knutson KL (2014) Sleep duration, quality, Schantz M (2016) Timing and quality of sleep
and timing and their associations with age in a in a rural Brazilian family-based cohort, the
community without electricity in Haiti. Am J Baependi Heart Study. Sci Rep 6:39283.
Hum Biol 26(1):80–86. https://doi.org/10. https://doi.org/10.1038/srep39283
1002/ajhb.22481 41. Ruiz FS, Beijamini F, Beale AD, Goncalves B,
37. Martins AJ, Vasconcelos SP, Skene DJ, Vartanian D, Taporoski TP, Middleton B, Krie-
Lowden A, de Castro Moreno CR (2016) ger JE, Vallada H, Arendt J, Pereira AC, Knut-
Effects of physical activity at work and life- son KL, Pedrazzoli M, von Schantz M (2020)
style on sleep in workers from an Amazonian Early chronotype with advanced activity
Extractivist Reserve. Sleep Sci 9(4):289–294. rhythms and dim light melatonin onset in a
https://doi.org/10.1016/j.slsci.2016.10.001 rural population. J Pineal Res 69(3):e12675.
38. Martins AJ, Isherwood CM, Vasconcelos SP, https://doi.org/10.1111/jpi.12675
Lowden A, Skene DJ, Moreno CRC (2020) 42. Samson DR, Manus MB, Krystal AD, Fakir E,
The effect of urbanization on sleep, sleep/ Yu JJ, Nunn CL (2017) Segmented sleep in a
wake routine, and metabolic health of residents nonelectric, small-scale agricultural society in
in the Amazon region of Brazil. Chronobiol Int Madagascar. Am J Hum Biol 29(4):e22979.
37(9–10):1335–1343. https://doi.org/10. https://doi.org/10.1002/ajhb.22979
1080/07420528.2020.1802287
Chapter 2
Abstract
Circadian rhythms are part of the body’s clock, which regulates several physiological and biochemical
variables according to the 24-h cycle. Ample evidence indicated disturbance of the circadian clock leads to
an increased susceptibility to several diseases. Therefore, a great effort has been made to find small
molecules that regulate circadian rhythm by high-throughput methods. Having crystal structures of core
clock proteins, makes them amenable to structure-based drug design studies. Here, we describe virtual
screening methods that can be utilized for the identification of small molecules regulating the activity of
core clock protein Cryptochrome 1.
Key words Circadian rhythm, Virtual screening, Cryptochrome, Docking, Drug discovery
1 Introduction
Guiomar Solanas and Patrick-Simon Welz (eds.), Circadian Regulation: Methods and Protocols,
Methods in Molecular Biology, vol. 2482, https://doi.org/10.1007/978-1-0716-2249-0_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
15
16 Seref Gul and Ibrahim Halil Kavakli
2 Materials
2.1 Homology If the protein does not have the crystal structure, homology mod-
Modeling Server eling tools should be used to construct 3D structure. Similarly,
missing atoms of the crystal structures can be added with the
same approach. Freely available and user-friendly homology mod-
eling servers can be used: RaptorX (http://raptorx.uchicago.edu/
StructPredV2/predict/), Swiss-Model (https://swissmodel.
expasy.org/interactive), and Phyre (http://www.sbg.bio.ic.ac.uk/
~phyre2/html/page.cgi?id¼index).
2.2 Programs to Pymol, Chimera, and VMD programs can be used to visualize and
Visualize Protein manipulate protein structures. Manual and tutorial of these pro-
Structures grams are available online to perform various operations such as
visualization, mutation, rotamer selection, and structure prepara-
tion for molecular dynamics (MD) simulations. Chimera and VMD
are free of charge for academic users with registration requirements
through their websites (https://www.cgl.ucsf.edu/chimera/down
load.html, https://www.ks.uiuc.edu/Development/Download/
download.cgi?PackageName¼VMD). Pymol offers an education
version for free and an alternatively open-source version can be
installed (https://pymol.org/dokuwiki/?id¼installation). The
usage of these programs will be mentioned in relevant sections.
2.3 Retrieving Crystal structures of CRY1 (PDB ID: 4K0R, 5T5X, or 6KX4) can
Protein Structures be downloaded from the protein data bank [33–35]. 4K0R is the
from Protein Data first resolved photolyase homology region (PHR) of mouse CRY1
Bank (PDB) structure with 2.65 Å in 2013 [33]. Higher-resolution PHR
domain of CRY1 structures with 1.84 and 2.00 Å were reported
recently [34, 35].
2.5 Small Molecules Commercially available small molecule (SM) libraries are available
in sdf format with 2D structural information. Since 3D structural
information and partial charges of atoms in molecules are required
for docking simulations, sdf format should be converted to pdbqt
format.
3 Methods
3.1 MD Simulation Two approaches are utilized for docking simulations in the litera-
ture: (a) docking molecules to the crystal structure of the receptor
and (b) docking molecules to the structure of the receptor equili-
brated under physiological conditions. We are not going to com-
pare the advantages and disadvantages of these approaches in this
chapter, but simply we prefer to use equilibrated receptor structures
in our docking simulations. Before starting, generate a new folder
named “structurebased,” download and save the below-mentioned
files into this folder.
3.1.1 Homology Since all crystal structures of CRY1 have missing atoms, homology
Modeling of CRY1 modeling should be performed. Swiss-Model server can be used to
get the full-length CRY1-PHR structure. The protein sequence of
mouse CRY1 can be retrieved from NCBI data bank.
1. Go to the website of NCBI (https://www.ncbi.nlm.nih.gov/),
select “Protein” from the drop-down menu and search for
“AAD39548.1”.
2. Click “FASTA” to get the fasta format of the CRY1 amino acid
sequence.
Structure-Based Drug Discovery for Clock Proteins 19
3.1.2 Preparing CRY1 for 1. To assign protonation states of residues by using PROPKA
MD Simulation server (http://server.poissonboltzmann.org/pdb2pqr) (see
Note 4).
(a) Click “Upload a PDB file” to upload CRY1.PDB.
(b) Select “Use PROPKA to assign protonation states at
provided pH.”
(c) Forcefield to use: CHARMM.
(d) Output naming scheme to use: CHARMM.
(e) In “Additional Options” check the following: “Ensure
that new atoms are not rebuilt too close to existing
atoms,” “Optimize the hydrogen bonding network,”
and “Add/keep chain IDs in the PQR file.”
(f) Download output “jobid.pqr”. Rename file as CRY1_p.
pqr.
(g) Open CRY1_p.pqr with PyMol, first open Pymol, then
File > Open > (browse molecule) > Open
File menu > Save molecule > check “one file” and
“global” and click OK > CRY1_p.pdb (into the “struc-
turebased” folder)
2. To solvate protein and add an appropriate concentration of salt
we are going to use VMD.
(a) Download top_all36_prot.rtf into the “structurebased”
folder from http://www.charmm-gui.org/?doc¼toppar
(see Note 5).
(b) First, we need to generate psf file. Generate a text file
“psfgen.in” and type commands given in Subheading 4.
Open VMD in the same directory with CRY1_p.pdb and
the topology file (in “structurebased” folder). In the
“VMD Main” panel
20 Seref Gul and Ibrahim Halil Kavakli
Fig. 1 Generating homology model of CRY1 using SWISS-MODEL server. (a) Query page, (b) template selection
page, (c) model page
Structure-Based Drug Discovery for Clock Proteins 21
resetpsf
mol delete all
source solvation.in
3.1.3 Running MD Before running the simulation, minimization of the system should
Simulation be performed. Then gradually heat and equilibrate the system.
Finally, we are going to use coordinates, velocity, and extended
system configuration files of minimized, heated, and equilibrated
system to run the MD simulation.
1. Following parameter files should be downloaded to working
directory (into the “structurebased” folder): par_all36_prot.
prm, par_all36_lipid.prm, par_all36_carb.prm, par_al-
l36_cgenff.prm from (http://www.charmm-gui.org/?
doc¼toppar) and toppar_water_ions_namd.str from (http://
mackerell.umar yland.edu/~kenno/cgenf f/program.
php#namd). The last file is the edited version of toppar_wa-
ter_ions.str to be able to use it in NAMD.
2. Make four folders and name them as Minimized, Heat, Equi-
librium, and Production. Next move parameter files given in
supplementary materials minimization.in, heating.in, equilib-
rium.in, production.in to corresponding folders.
3. Go to “Minimize” folder and run minimize.in via NAMD by
typing the following:
3.2 Docking Determining the target pocket, preparing molecules and receptor
(s) in pdbqt format are crucial steps in docking studies. Below we
are going to describe these steps for CRY1. Before going into
details generate a folder named “docking.” Move CRY1.pdb file
to the docking folder.
3.2.1 Preparation of 1. Open Autodock Tools program from the terminal, e.g., ~/
CRY1 for Docking MGLTools-1.5.6/MGLToolsPckgs/AutodockTools then
Simulations File > Rad Molecule > CRY1.pdb
Grid > Macromolecule > Choose > CRY1 > Select
molecule
New panel should appear: type CRY1.pdbqt > Save (see
Note 12).
2. A grid center point and box size should be determined for
docking simulations. Since the primary pocket of CRY1 is
targeted, coordinates of the side chain terminal carbon atom
of Arg358 (CZ in pdb file) can be used as grid center. Then, fine
tune the center by using the “Grid Options” panel. To visualize
the target pocket, the grid box panel should be used as follows:
Grid > Grid Box > number of points in x-dimension: 18;
y-dimension: 20; z-dimension: 20; x center: 11.7; y center:
35.7; z center: 16.4 (Fig. 3) (see Note 13).
3. Target pocket size and center information determined in step
2 will be provided in the configuration file that includes the
following keywords:
receptor, out, center_x, center_y, center_z, size_x, size_y,
size_z. Although these parameters are self-explanatory, options
that can be specified in AutodockVina are explained in notes
(see Note 14).
4. A sample configuration file named as “cry1_conf.txt” is
provided below:
24 Seref Gul and Ibrahim Halil Kavakli
Fig. 3 CRY1.pdbqt is visualized using AutodockTools. Grid box is colored in red-green-blue in x, y, and
z directions, respectively
receptor = CRY1.pdbqt
center_x = -11.7
center_y = 35.7
center_z = -16.4
size_x = 18.0
size_y = 20.0
size_z = 20.0
3.2.2 Preparation of Small molecule libraries from different companies are freely avail-
Small Molecules for able and can be downloaded from their websites. One of the
Docking Simulations commonly used libraries is ZINC library that has many tranches
classified according to properties of molecules such as molecular
weight and LogP. In addition to these physical properties, mole-
cules are under predefined categories, e.g., fragments, lead-like,
and drug-like.
1. Go to website: https://zinc.docking.org/tranches/home/
Structure-Based Drug Discovery for Clock Proteins 25
~/MGLTools-1.5.6./MGLToolsPckgs/AutoDockTools/Utilities24/
prepare_ligand4.py -l sm1.pdb
3.2.3 Running and Our receptor (CRY1.pdbqt) and ligands (sm1.pdbqt and others)
Analyzing AutoDock Vina are ready for docking simulations. We are going to use Autodock-
Simulations Vina software to run simulations. This program does not need to be
installed. Once the program is downloaded, giving a path to the
“vina” executable will be sufficient to run the program. “cry1_conf.
txt” was given in step 4 of Subheading 3.2.1. If all SMs and CRY1.
pdbqt is in the same folder,
1. Type following to the terminal:
4 Notes
Output (optional):
Misc (optional):
Information (optional):
obabel -H
~/MGLTools-1.5.6./MGLToolsPckgs/AutoDockTools/Utilities24/
prepare_ligand4.py
python2.7 vina_screen_get_top.py 10
References
1. Xie YL, Tang QM, Chen GJ, Xie MR, Yu SL, transcription and metabolism. Cell Metab
Zhao JJ, Chen LL (2019) New insights into 25(4):961. https://doi.org/10.1016/j.cmet.
the circadian rhythm and its related diseases. 2017.03.019
Front Physiol 10:ARTN 682. https://doi.org/ 10. Zhang R, Lahens NF, Ballance HI, Hughes
10.3389/fphys.2019.00682 ME, Hogenesch JB (2014) A circadian gene
2. Gachon F, Nagoshi E, Brown SA, Ripperger J, expression atlas in mammals: implications for
Schibler U (2004) The mammalian circadian biology and medicine. Proc Natl Acad Sci U S
timing system: from gene expression to physi- A 111(45):16219–16224. https://doi.org/
ology. Chromosoma 113(3):103–112. 10.1073/pnas.1408886111
https://doi.org/10.1007/s00412-004- 11. Cal-Kayitmazbatir S, Kulkoyluoglu-Cotul E,
0296-2 Growe J, Selby CP, Rhoades SD, Malik D,
3. Kavakli IH, Sancar A (2002) Circadian photo- Oner H, Asimgil H, Francey LJ, Sancar A, Kru-
reception in humans and mice. Mol Interv ger WD, Hogenesch JB, Weljie A, Anafi RC,
2(8):484–492. https://doi.org/10.1124/mi. Kavakli IH (2020) CRY1-CBS binding regu-
2.8.484 lates circadian clock function and metabolism.
4. Kyriacou CP, Hastings MH (2010) Circadian FEBS J. https://doi.org/10.1111/febs.15360
clocks: genes, sleep, and cognition. Trends 12. Toh KL, Jones CR, He Y, Eide EJ, Hinz WA,
Cogn Sci 14(6):259–267. https://doi.org/ Virshup DM, Ptacek LJ, Fu YH (2001) An
10.1016/j.tics.2010.03.007 hPer2 phosphorylation site mutation in familial
5. Kavakli IH, Baris I, Tardu M, Gul S, Oner H, advanced sleep phase syndrome. Science
Cal S, Bulut S, Yarparvar D, Berkel C, 291(5506):1040–1043
Ustaoglu P, Aydin C (2017) The photolyase/ 13. Vanselow K, Vanselow JT, Westermark PO,
cryptochrome family of proteins as DNA repair Reischl S, Maier B, Korte T, Herrmann A,
enzymes and transcriptional repressors. Photo- Herzel H, Schlosser A, Kramer A (2006) Dif-
chem Photobiol 93(1):93–103. https://doi. ferential effects of PER2 phosphorylation:
org/10.1111/php.12669 molecular basis for the human familial
6. Takahashi JS (2017) Transcriptional architec- advanced sleep phase syndrome (FASPS).
ture of the mammalian circadian clock. Nat Rev Genes Dev 20(19):2660–2672. https://doi.
Genet 18(3):164–179. https://doi.org/10. org/10.1101/gad.397006
1038/nrg.2016.150 14. Xu Y, Toh KL, Jones CR, Shin JY, Fu YH,
7. Hirano A, Yumimoto K, Tsunematsu R, Ptacek LJ (2007) Modeling of a human circa-
Matsumoto M, Oyama M, Kozuka-Hata H, dian mutation yields insights into clock regula-
Nakagawa T, Lanjakornsiripan D, Nakayama tion by PER2. Cell 128(1):59–70. https://doi.
Keiichi I, Fukada Y (2013) FBXL21 regulates org/10.1016/j.cell.2006.11.043
oscillation of the circadian clock through ubi- 15. Takano A, Uchiyama M, Kajimura N,
quitination and stabilization of cryptochromes. Mishima K, Inoue Y, Kamei Y, Kitajima T,
Cell 152(5):1106–1118. https://doi.org/10. Shibui K, Katoh M, Watanabe T,
1016/j.cell.2013.01.054 Hashimotodani Y, Nakajima T, Ozeki Y,
8. Yoo SH, Mohawk JA, Siepka SM, Shan Y, Huh Hori T, Yamada N, Toyoshima R, Ozaki N,
SK, Hong HK, Kornblum I, Kumar V, Okawa M, Nagai K, Takahashi K, Isojima Y,
Koike N, Xu M, Nussbaum J, Liu X, Chen Z, Yamauchi T, Ebisawa T (2004) A missense var-
Chen ZJ, Green CB, Takahashi JS (2013) iation in human casein kinase I epsilon gene
Competing E3 ubiquitin ligases govern circa- that induces functional alteration and shows
dian periodicity by degradation of CRY in an inverse association with circadian rhythm
nucleus and cytoplasm. Cell 152(5): sleep disorders. Neuropsychopharmacology
1091–1105. https://doi.org/10.1016/j.cell. 29(10):1901–1909. https://doi.org/10.
2013.01.055 1038/sj.npp.1300503
9. Krishnaiah SY, Wu G, Altman BJ, Growe J, 16. Weitzman ED, Czeisler CA, Coleman RM,
Rhoades SD, Coldren F, Venkataraman A, Spielman AJ, Zimmerman JC, Dement W,
Olarerin-George AO, Francey LJ, Richardson G, Pollak CP (1981) Delayed
Mukherjee S, Girish S, Selby CP, Cal S, Ubey- sleep phase syndrome. A chronobiological dis-
dullah ER, Sianati B, Sengupta A, Anafi RC, order with sleep-onset insomnia. Arch Gen
Kavakli IH, Sancar A, Baur JA, Dang CV, Psychiatry 38(7):737–746
Hogenesch JB, Weljie AM (2017) Clock regu- 17. Katzenberg D, Young T, Finn L, Lin L, King
lation of metabolites reveals coupling between DP, Takahashi JS, Mignot E (1998) A CLOCK
Another random document with
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stolen away under cover of the night and disappeared, rather than
face an investigation.
The daily papers had blazoned abroad the shooting of Randall
Batterly, and the subsequent trial of Wanza Lyttle, and my name had
appeared in the account, the writer who was my father’s lawyer
explained. A letter to the postmaster at Roselake had resulted in
further establishing my identity.
The writer had the honor to inform me that my father had left a snug
little fortune—the result of some recent fortunate mining ventures—
that would accrue to me, and he begged me to come back to my
southern home and take my rightful place among the people. I shook
my head at this. Who was there in the old home who would welcome
me? My mother was long since dead—my father gone. There was
no one belonging to me left in the old place. It would be more
strange and forlorn than an entirely new community. I should like to
visit it again. But that was all.
I dropped the letter to the floor, and sat thinking of Haidee. And as I
thought I smiled tenderly. After a time I decided that Haidee should
see these important letters—that I should go to her. And on a sudden
impulse I rose up.
As I opened the door the snow was falling, and there was a ring
around the moon. I left the door open and stepped back into the
house, going to the cedar room to get my sweater. When I returned,
a woman with snow-powdered hair was stepping hesitatingly across
the threshold. Haidee!
“It is you! Out so late—alone!” I began. “And in this storm.”
But the big eyes only smiled at me, and she stood there like a
beautiful wraith in her long gray cloak.
“Let me take your cloak,” I said.
I went to her, and she put both hands on my shoulders impulsively.
“I haven’t thought of the weather. Ever since I saw you last I’ve
thought of you,—and thought, and thought. It’s Christmas Eve, you
know. I have come to wish you a Merry Christmas, and I have
brought you a Christmas gift—one to keep till spring, at least.”
“Come to the fire,” I urged.
She sat down and I sat down opposite her. The firelight caressed
her, played in her eyes, ruddied her cheeks that were glowing from
her walk through the wintry air.
“In all the time I have known you this is the first time I have ever
shared your fire,” she whispered.
There was a silence. I could hear my heart-beats. How fine of her to
come to me in this womanly fashion! I sat and watched her. A lock of
hair had fallen over her ivory brow. She had dropped her head
forward on to her hand, and her dewy lips were parted. I stooped
closer, closer still. A tear slipped down on her smooth cheek and
glistened in the firelight as I gazed. She turned her face away.
“What gift have you brought me?” I whispered.
There was a movement in the shadows beyond the circle of light
cast by the green-shaded lamp—a rustle and a stir—then a swift
hurtling of a small lithe figure across the open space—a pause—a
swooping, frantic clutch of young strong arms about my neck, and
Joey, all wet and steaming in his snowy coat, had me fast, shouting
in my ear, over and over again:
“I’m your Christmas gift, Mr. David! I’m your Christmas gift.”
He was in my arms, and Haidee had drawn back and was smiling at
me, her eyes like great luminous pools of fire.
“What a wonderful, wonderful present,” I responded shakily. “Now,
who could have sent me this very best present in the world?”
“Bell Brandon,” shrieked my little lad. “She did not send me—she
brought me.”
“Then—she must have another gift for me,” I said boldly, and held
out my hand to Haidee.
She shook her head, her eyes grave, but her lips still smiling.
“I have brought Joey to you—but—I cannot stay. I am going away.
Will you keep my boy until I return?”
“You are going away?”
She bent her head.
“I am going to take Wanza back East. I want to go away for a time—
it is best for me to go. But—you must not be separated from Joey all
this long winter, David Dale. My boy shall stay with you—and in the
spring I shall come for him—or come back to stay at Hidden Lake.”
“You are going away—soon—after Christmas?”
“To-morrow. We are going to-morrow—Wanza and I—we decided it
only to-day. I have some matters to attend to in New York. I must go
at once.”
“Christmas Day?”
“Yes.”
“Wait—do not go—stay with me as my wife, my wife! I have sold my
book—I am free too, of an old, old shadow. Oh, I have much to tell
you—much to talk over with you. Wait—let me read to you some
letters.”
My voice was rough with emotion. She held up her hand.
“When I come back, David Dale, my friend—not now. We need to
gain perspective—you and I. I have been through an ordeal—I am
shaken—I am not myself. I don’t see clearly. And as for you—David
Dale, there is much for you to learn.”
“What do you mean?” I cried brusquely.
She smiled at me sweetly and a little sadly.
“Oh, you are a stupid blundering David.” She shook her head. “But—
wait till spring.”
“There is so much I want to say—explain,” I stammered.
“Wait till spring.”
“But I cannot keep Joey. I cannot let you go without your boy.”
“He will be better off with you.”
“I cannot accept such a sacrifice.”
On this point I remained firm. We argued. Haidee entreated, and
Joey begged to be allowed to stay. I would not listen to either voice. I
arose at last.
“Joey,” I said, speaking slowly, in order to steady my voice, “I have
one more bolt to put in the sled I am making for you. Will you come
to the workshop with me?”
And in the shop away from every eye, I said good-bye to my lad. And
as I kissed him the old doubt stirred. Was I so sure he was Haidee’s
child?
Old Lundquist came for Haidee; and we said a conventional good-
bye beneath his prying eyes.
Until twelve I waited and watched for Wanza, expecting every instant
to hear Captain Grif’s voice at the door, and to see Wanza step over
the threshold. Surely she would not go without some last word to me.
But she came not.
CHAPTER XXIV
“THE FLOWER WILL BLOOM ANOTHER YEAR”
I SAT by my fire throughout the long night. When dawn came I rose,
went to the door and threw it wide and stepped outside into the
unstained air of the morning. There was a carpet of snow on the
ground, the bushes were like gleaming teepes, and the limbs of the
pine trees were weighted with icicles. I repeated to myself Thoreau’s
words: “God exhibits himself in a frosted bush to-day, as much as he
did in a burning one to Moses.”
The light was purple and cold and solemn, the moon still hung in the
gray of the western sky, but in the East there was a glorious band of
crimson and the mountain tops looked as if aflame with little bonfires.
As I stood there a ruby-crowned kinglet fluttered from twig to twig of
the elderberry bush hard by, emitting its bright “zei, zei,” and a
chickadee answered with a merry “chickadee-a-dee, dee, dee,” from
the yew grove. I waited. I was praying the kinglet would sing. And
presently the tiny thing began. It poured forth its strong sweet notes
in a succession of trills.
“Bird,” I said, “you are a wonder. I know that the muscles in your
throat are almost microscopic. I have always told Joey—” But here I
ceased to admonish the bird, I went back up the porch steps.
As I was closing the door I heard the rattle of the stage as it passed
along the river road on its way to the village. The driver shouted a
merry Christmas to some one on the road. I threw a fresh log on the
fire and sat down heavily in my chair. It was Christmas morning—
and they had gone!
I drowsed after a time, lying back in my great chair with the collie
asleep at my feet. When I awakened the sun was high, and the world
outside my window was so sparkling and bright that it dazzled my
sight. I went to the kitchen, kindled a fire, and opened the kitchen
door to let the collie out. I was washing my hands at the wash-bench
in the corner, when I heard the latch of the door click. Footsteps
crossed the floor, some one was coming up behind me saying:
“I have brought a chicken pie for your dinner, Mr. Dale—Dad’ll be
along soon—and I wish you a Merry Christmas.”
It was Wanza.
She stood there as she had so often stood before, a white-covered
basket on one arm, the other filled with bundles. But her face was
pale to-day, and her glorious hair was swept straight back from her
brow and tucked away beneath a net, and her apparel was sober
gray. I stared at her and stared and stared, until the pink ran up in
her cheek and she dropped the bundles and set down the basket,
that she might put her hands over her abashed face. I stood there
and felt shaken and dumbfounded, not attempting to speak, afraid
indeed of the sound of my own voice.
The fire crackled. Cheerily through the door Wanza had left open
behind her, came the chickadee’s note. The sunlight was dazzling as
it struck into my eyes from the white oilcloth on the kitchen table. The
room seemed suddenly illumined, the air electric and revitalized. At
length I stammered out:
“Thank you, thank you!”
“It’s only chicken pie,” she whispered.
“Thank you for not going.”
At that she threw up her head, her hands dropped. She said proudly:
“Did you think I’d go on Christmas Day? Did you think I’d have the
heart to go, Mr. Dale?”
“Yes,” I said wearily, “I thought you had gone, Wanza. Why not?”
“And I’ll tell you why not! It’s because you decided Joey was to go
that I could not go. I could not go and leave you when I found Joey
was to go—oh, no!”
“But you must go some day, Wanza,” I said, scarce knowing what I
said.
“And why must I go some day? Why must I? I tell you what I’m going
to do, Mr. David Dale, I’m going to stay on here in Roselake, and I
am going to live up to the very best there is in me. I am going to
improve and grow big and fine and womanly. I’m going to do it right
here. And then maybe some day,” she sighed, “when Dad does not
need me any more, and you do not need me any more, I will have
enough money saved up, and I will go away and get educated.”
In her excitement she had pressed closer to me and laid one hand
against my chest. I placed my own hand over it as I said very gently:
“Let me teach you, Wanza—be my pupil. I will become your tutor in
earnest, if you will have me. Yes! I will go to your father’s house
every day to instruct you,—and it will give me great happiness. Ah,
Wanza, now that Joey has gone I feel so futile—so useless! Let me
undertake your education, child.”
The burning eyes came up to mine, and questioned them. The pale
face flushed. There was a pathetic tremulousness about the lips.
“Say yes,” I urged.
Her head drooped, lowered itself humbly until her hair brushed my
arm, and suddenly she kissed my hand, passionately, gratefully. “Oh,
Mr. David Dale,” she breathed, “you’re grand! That’s what you are.
Yes and yes, and yes!”
And so I ate my dinner with Wanza and Captain Grif sitting opposite
me at the table, and Wanza flouted me when I would have served
her too liberally with the most succulent bits of the pie, and Captain
Grif rallied me when I confessed that I had small appetite, and
produced a bottle of root beer and a bag of cheese cakes from the
basket.
Night came down at last to my weary soul and soon after it grew
dark Wanza and her father departed. I locked the door behind them
and I threw myself, dressed as I was, on my bunk and buried my
head in the pillows. The evening wore on. The fire sputtered and
burned low, the wind came up and hissed around the cabin. A coyote
howled from some distant hill. The room grew dark. A pall was on my
heart.
As the winter wore on I became vastly interested in Wanza’s
education. I gave two hours each day to her lessons. And not many
evenings passed without lessons in the snug little room beneath the
eaves of the cottage she called home. There with our books open
before us, beneath the light from the swinging lamp, we pored over
tedious pages shoulder to shoulder, smiled on by old Grif and
encouraged by Father O’Shan, who ofttimes shared our evenings.
It was wonderful the improvement I marked in Wanza as the weeks
slipped past. Her English improved markedly. She was painstaking
and indefatigable. She applied herself so assiduously that I began to
fear lest she should overwork, as the warm spring days came on.
“Don’t study too hard,” I cautioned her one day.
“I can’t study too hard,” she flashed back at me. And then she
smiled. But I knew she was terribly in earnest.
It was that same day that Father O’Shan quoted to me, as we were
walking along the river road together:
“Shed no tear—Oh, shed no tear!
The flower will bloom another year.
Weep no more—Oh, weep no more!
Young buds sleep in the root’s white core.”
“Do you mean that for me, Father?” I asked.
“For you—yes. And many like you.”
My heart swelled. I looked about me. Buttercups were gilding the sod
—the pussy willows were in bloom along the river. It was the spring.
I went home and raked the dead leaves and pine needles away from
under the trees in the Dingle. A few yellow violets were springing up.
From beyond the syringa thicket a faint “witchery, witchery, witchery,”
greeted my ears.
I went forward cautiously. Peering through the interlaced branches I
saw the songster. He was swinging on a thorn bush, a wonderfully
brilliant little chorister in his black cap and yellow stole. I whistled. He
cocked his head on one side, fixed me with his bright eye, then flew
to a willow tree and favored me with another burst of song. This time
he seemed to oft repeat, “Which way, oh?” He sang it so persistently
that presently I replied, “Straight on, sir.”
I went to the cabin and consulted the calendar. It was the last day of
March.
My spirit, that had seemed earthward crushed for months, grew
lighter in the sweet spring days that followed. I took the return of
April as a long-fore-gone right. I ploughed and planted, I made bird
houses and arranged bird-baths in the groves hard by the cabin. I
paddled in my canoe on the river, and fished in the adjacent creeks.
And I went with Wanza through the woods on many a trillium hunt.
Sometimes almost to breathlessness I felt Wanza’s charm, the
galvanism she could always transmit to those with her intensified by
some new strange quality I could not name. It was like a fillip given
my dispassion. When she laughed and chirped to the squirrels, when
she carried a wounded bird in her breast, when she stood on tip-toe,
her face like a taper-flame, to greet the whole outdoors with wide-
flung arms, I caught my lip between my teeth and watched her with
observant eyes. Her beauty grew. Even Father O’Shan remarked it.
The gowns of pink she wore once served to deepen the rose tint in
her fair cheeks; but her cheeks needed no such service now; they
were like a red-rose heart. She had taken to smoothing and banding
her hair and twisting it back behind her small ears with big shell pins.
Her head seen thus was as lovely a shape as any Greuze ever
painted. She frequently wore thin blouses of white, and I seldom saw
her feet in sandals—she had a sleeveless black gown that she wore
to a country dance one evening when I was her escort. Looking at
her that night I could scarcely believe it was Wanza, my old friend
and playmate whom I was in attendance upon, and I paid her some
rather silly compliments and was promptly rebuked for my gallantry.
It was a tidy enough fortune my dear old father had left me. I had
been able to do many things to make Wanza and Captain Grif
comfortable and happy during the long winter. Among other things I
had purchased a piano for Wanza to replace the old melodeon, and
delighted Captain Grif with the gift of a phonograph. And last, but not
least, I had made the last payment on the little cottage in which they
lived and presented the deed to Captain Grif on his sixty-fifth
birthday.
Dear Captain Grif! His manner of accepting this last gift was
characteristic.
“Tain’t for myself I’d take it. I’d just about as lief worry along and save
and scrimp toward makin’ the final payment— I ’low I’d sooner; I like
the glory, and when you have a soft thing handed to you there ben’t
nothin’ achieved. I’m meanin’ it, s-ship-mate. Things we earn is the
things we ’preciate. But I take it kindly of you. And for Wanza’s sake I
thank you and accept. ’Tis hard on the gal—pinchin’ and scrimpin’—
and peddlin’ in winter is about played out—the roads is in bad shape
for gettin’ about, you’ll ’low. Now with the house paid for, the gal’ll
have what she earns for ribbons and furbelows and trinkets. And
ownin’ sech a face as hern, Mr. Dale—though it don’t need no
adornin’—sure makes a gal long for fixin’s. I’m grateful and pleased
for her sake—I sure be.” Tears dimmed his kind old eyes. His hand
came out to me. “Shake hands, David Dale, man; you’re a friend—a
friend. We need friends—the gal and I—seems like we need ’em
more’n we used since all we been through,—and I want to say right
here that Wanza never would’a perked up if it hadn’t a been for your
helpin’ her this winter. She was pretty well down, Wanza was. Well,
in my youth, young folks was different. I used to think—I used to
think one time—well, there, by golly, s-ship-mate, it makes no
difference what I used to think! I was mistook, I ’low. It sure is great
for a man and gal to be such friends as you and Wanza—no
foolishness—no tomfoolery!—it’s unusual—I ain’t sayin’ that it tain’t
—but it’s fine, s-ship-mate, it’s fine.”
“I’M GRATEFUL AND PLEASED”
Through the winter I had had frequent letters from Haidee—frank,
friendly letters, filled with stories of Joey—and a few printed epistles
from the lad; one in particular that impressed me; “Joey is all rite,” it
said.
I discussed this with Wanza, who said tearfully:
“His saying that makes me think he isn’t. He is such a plucky little
chap. He would not have you worrying. Not that I think he’s sick—
sure enough sick, you know; but I just feel sure he’s pining.”
“Please—please, Wanza, don’t put that thought into my mind,” I said
hastily. “If I thought Joey were happy I could more easily bear his
absence.”
She looked at me and shook her head. Then she smiled.
“He’ll do well enough till spring. But he will be counting the days, all
right.”
CHAPTER XXV
MY SURPRISE