The biofilm growth mode is important in both the intestinal and environmental phases of the Vibrio cholerae life cycle. Regulation of biofilm formation involves several transcriptional regulators and alternative sigma factors. One such factor is the alternative sigma factor RpoN, which positively regulates biofilm formation. RpoN requires bacterial enhancer-binding proteins (bEBPs) to initiate transcription. The V. cholerae genome encodes seven bEBPs (LuxO, VC1522, VC1926 [DctD-1], FlrC, NtrC, VCA0142 [DctD-2], and PgtA) that belong to the NtrC family of response regulators (RRs) of two-component regulatory systems. The contribution of these regulators to biofilm formation is not well understood. In this study, we analyzed biofilm formation and the regulation of vpsL expression by RpoN activators. Mutants lacking NtrC had increased biofilm formation and vpsL expression. NtrC negatively regulates the expression of core regulators of biofilm formation (vpsR, vpsT, and hapR). NtrC from V. cholerae supported growth and activated glnA expression when nitrogen availability was limited. However, the repressive activity of NtrC toward vpsL expression was not affected by the nitrogen sources present. This study unveils the role of NtrC as a regulator of vps expression and biofilm formation in V. choleraeIMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, contributing to both environmental survival and transmission to a human host. Identifying key regulators of V. cholerae biofilm formation is necessary to fully understand how this important growth mode is modulated in response to various signals encountered in the environment and the host. In this study, we characterized the role of RRs that function as coactivators of RpoN in regulating biofilm formation and identified new components in the V. cholerae biofilm regulatory circuitry.

Lamins A and C (products of the LMNA gene) are found in roughly equal amounts in peripheral tissues, but the brain produces mainly lamin C and little lamin A. In HeLa cells and fibroblasts, the expression of prelamin A (the precursor to lamin A) can be reduced by miR-9, but the relevance of those cell culture studies to lamin A regulation in the brain was unclear. To address this issue, we created two new Lmna knock-in alleles, one (Lmna(PLAO-5NT)) with a 5-bp mutation in a predicted miR-9 binding site in prelamin A's 3' UTR, and a second (Lmna(PLAO-UTR)) in which prelamin A's 3' UTR was replaced with lamin C's 3' UTR. Neither allele had significant effects on lamin A levels in peripheral tissues; however, both substantially increased prelamin A transcript levels and lamin A protein levels in the cerebral cortex and the cerebellum. The increase in lamin A expression in the brain was more pronounced with the Lmna(PLAO-UTR) allele than with the Lmna(PLAO-5NT) allele. With both alleles, the increased expression of prelamin A transcripts and lamin A protein was greater in the cerebral cortex than in the cerebellum. Our studies demonstrate the in vivo importance of prelamin A's 3' UTR and its miR-9 binding site in regulating lamin A expression in the brain. The reduced expression of prelamin A in the brain likely explains why children with Hutchinson-Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease.

Background

Abnormal frontal plane gait mechanics are known risk factors for knee osteoarthritis, but their role in early cartilage degeneration after anterior cruciate ligament reconstruction (ACLR) is not well understood. Hypothesis/Purpose: The objective was to evaluate the association of frontal plane gait mechanics with medial knee cartilage magnetic resonance (MR) relaxation times over 1 year in patients with ACLR and controls. It was hypothesized that (1) there will be an increase in frontal plane medial knee loading and medial knee MR relaxation times over time in the patients with ACLR, and (2) increases in frontal plane medial knee loading will be associated with an increase in medial knee MR relaxation times.

Study design

Case-control study; Level of evidence, 3.

Methods

Patients with ACLR (n = 37) underwent walking gait analyses and bilateral quantitative MR imaging (MRI) before surgery and at 6 and 12 months after ACLR. Healthy control participants (n = 13) were evaluated at baseline and 12 months. Gait variables included peak knee adduction moment (KAM), KAM impulse, and peak knee adduction angle. MRI variables included medial femur and medial tibia whole compartment and subregional T_1ρ and T₂ relaxation times. Statistical analyses included a comparison of changes over time for gait and MRI variables, correlations between changes in gait and MRI variables over time, and differences in change in MRI variables in patients who showed an increase versus decrease in KAM impulse.

Results

There were significant increases in medial T_1ρ (Δ 4%-11%) and T₂ (Δ 2%-10%) relaxation times from baseline to 6 months for both knees in the ACLR group and in KAM (Δ 13%) for the injured knee. From baseline to 6 months, patients who had an increase in KAM impulse in the injured knee had a greater increase in medial T_1ρ and T₂ relaxation times as compared with those who did not have an increase in KAM impulse. Longitudinal changes for the control group were not significant.

Conclusion

There is an increase in medial knee relaxation times over the first 6 months after ACLR. People with an increase in medial knee loading show an increase in medial knee relaxation times when compared with those who do not have an increase in medial knee loading over the first 6 months.

Objective

In postmenopausal black women in the Women's Health Initiative randomized trial, estrogen alone reduced breast cancers but its comprehensive influence on health outcomes in black women is unknown. Therefore, we examined this issue in the Women's Health Initiative overall and by African ancestry.

Methods

A total of 1,616 black women with prior hysterectomy, including 1,061 with percent African ancestry determination, at 40 US centers were randomly assigned to conjugated equine estrogen (0.625 mg/d) or placebo for 7.2 years' (median) intervention with 13 years' cumulative follow-up. Coronary heart disease (CHD) and breast cancer were primary efficacy and safety outcomes, respectively. A global index also included stroke, colorectal cancer, hip fracture, pulmonary embolism, and death.

Results

Black women in the estrogen-alone group compared with black women in the placebo group had fewer breast cancers (17 vs 40, hazard ratio [HR] 0.47, 95% CI 0.26-0.82). In women with more than 80% African ancestry, breast cancer HR was lower (0.32, 95% CI 0.12-0.86, trend P = 0.04 for ancestry effect). Most other outcomes including CHD, stroke, hip fracture, and the global index were null with estrogen use in black women; a global index effect was more favorable in younger black women (HR 0.65, 95% CI 0.43-0.98).

Conclusions

In black postmenopausal women with prior hysterectomy, estrogen alone significantly reduced breast cancer incidence with no adverse influence on CHD, venous thromboembolism, or all-cause mortality. Favorable estrogen-alone global index effects in younger black women warrant further study.

Nanoscale secondary ion MS (NanoSIMS) imaging makes it possible to visualize stable isotope-labeled lipids in cells and tissues at 50 nm lateral resolution. Here we report the use of NanoSIMS imaging to visualize lipids in mouse cells and tissues. After administering stable isotope-labeled fatty acids to mice by gavage, NanoSIMS imaging allowed us to visualize neutral lipids in cytosolic lipid droplets in intestinal enterocytes, chylomicrons at the basolateral surface of enterocytes, and lipid droplets in cardiomyocytes and adipocytes. After an injection of stable isotope-enriched triglyceride-rich lipoproteins (TRLs), NanoSIMS imaging documented delivery of lipids to cytosolic lipid droplets in parenchymal cells. Using a combination of backscattered electron (BSE) and NanoSIMS imaging, it was possible to correlate the chemical data provided by NanoSIMS with high-resolution BSE images of cell morphology. This combined imaging approach allowed us to visualize stable isotope-enriched TRLs along the luminal face of heart capillaries and the lipids within heart capillary endothelial cells. We also observed examples of TRLs within the subendothelial spaces of heart capillaries. NanoSIMS imaging provided evidence of defective transport of lipids from the plasma LPs to adipocytes and cardiomyocytes in mice deficient in glycosylphosphatidylinositol-anchored HDL binding protein 1.

The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated "reciprocal knock-in mice"-mice that make lamin B2 from the Lmnb1 locus (Lmnb1(B2/B2)) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2(B1/B1)). Lmnb1(B2/B2) mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1(B2/B2) mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2(B1/B1) mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other.

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5-25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL-GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.