HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors.

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants in vivo.

Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env.

VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.

Risk of readmission for injury in patients with epilepsy in the United States - A population-based study.

OBJECTIVE: The objective of this study was to determine the 30-day injury readmission risk among persons with epilepsy vs. without epilepsy using a nationally representative US database. Secondary objectives were to examine the factors associated with injury-related readmissions among those with epilepsy and identify specific causes of readmissions within 30 days of index admission. METHODS: Hospitalized individuals of all ages with epilepsy as the primary diagnosis were identified using validated International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes in the 2014 Nationwide Readmission Database (NRD). Primary outcome was 30-day readmission for an injury defined by ICD-9-CM diagnosis codes following discharge from index hospitalization. Subgroup differences in the groups with epilepsy and without epilepsy were estimated using standardized mean difference scores that are calculated with means and variances of the covariates. Multinomial logistic regressions were conducted to determine the 30-day injury readmission risk and examine the factors associated with injury-related readmissions. RESULTS: There were 60,074 unique persons with epilepsy (mean age: 42.53 years, female: 49.32%) and 9,282,952 without epilepsy (mean age: 44.46 years, female: 59.43%). A higher proportion of persons with epilepsy (n = 215, 0.34%) vs. without epilepsy (n = 22,783, 0.22%) had a 30-day readmission due to an injury. After adjusting for covariates, persons with epilepsy had higher odds of 30-day readmission due to an injury (adjusted OR: 1.40, 95% CI: 1.20-1.62, p < 0.0001). Factors associated with an injury-related readmission in persons with epilepsy include the following: increasing age (OR: 1.01, 95% CI: 1.00-1.02, p = 0.02), transfer to short term hospital/other facility (OR: 1.50, 95% CI: 1.00-2.27, p = 0.05), discharged against medical advice/discharge destination unknown (OR: 2.26, 95% CI: 1.40-4.45, p = 0.02), and higher Elixhauser comorbidity index (OR: 1.02, 95% CI: 1.01-1.03, p < 0.0001). CONCLUSIONS: Higher odds of 30-day injury readmissions were observed in persons with epilepsy vs. without epilepsy. Optimizing the management of comorbid conditions during the patient's index admission for epilepsy, minimizing discharges against medical advice, and fostering outreach programs to those who have been transferred to short-term hospitals or facilities may reduce 30-day readmissions due to an injury.

The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles.

InDrosophila, a transcriptional feedback loop that is activated by CLOCK-CYCLE (CLK-CYC) complexes and repressed by PERIOD-TIMELESS (PER-TIM) complexes keeps circadian time. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control PER, TIM, and CLK levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. To identify clock-relevant phosphatases, clock-cell-specific RNAi knockdowns ofDrosophilaphosphatases were screened for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons whenLarexpression is knocked down during development, but not in adults. Loss ofLarfunction eliminates sLNvdorsal projections, but PDF expression persists in sLNvand large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF,LarRNAi knockdown flies anticipate the lights-on and lights-off transition normally. Our results demonstrate thatLaris required for sLNvdorsal projection development and suggest that PDF expression in LNvcell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle. SIGNIFICANCE STATEMENT: In animals, circadian clocks drive daily rhythms in physiology, metabolism, and behavior via transcriptional feedback loops. Because key circadian transcriptional activators and repressors are regulated by phosphorylation, we screened for phosphatases that alter activity rhythms when their expression was reduced. One such phosphatase, leukocyte-antigen-related (LAR), abolishes activity rhythms, but does not disrupt feedback loop function. Rather,Lardisrupts clock output by eliminating axonal processes from clock neurons that release pigment-dispersing factor (PDF) neuropeptide into the dorsal brain, but PDF expression persists in their cell bodies and remaining projections. In contrast to flies that lack PDF, flies that lackLaranticipate lights-on and lights-off transitions normally, which suggests that the remaining PDF expression mediates activity during light/dark cycles.

Closure of chronic non healing ankle ulcer with low level laser therapy in a patient presenting with thalassemia intermedia: Case report.

In this single case study, the possible effect of low-level laser therapy (LLLT) was explored in the form of light emitting diodes on a chronic non-healing wound of 6 months duration in an 18-year-old male patient suffering from thalassemia intermedia. After irradiation, with LLLT dosage of 17.3 J/cm(2) for 8 min for 2 weeks duration followed by proliferative dosage of 8.65-4.33 J/cm(2) for 4 min from 3(rd) week to 6(th) week for 2 min along with antibiotics vancomycin (15 mg/kg) and a combination of amoxicillin and clavulanic acid (1 g). Proliferation of healthy granulation tissue was observed with decrease in score of pressure ulcer scale with complete re-epithelialization eventually LLLT irradiation could be a novel method of treatment for chronic non-healing wound in a thalassemia intermedia patient and an useful adjunct to standard care of treatment of pressure ulcers. It is postulated that LED irradiation augments wound healing with an early closure and no recurrence at the irradiated site even after follow up of 6 months.

Priming locomotor training with transspinal stimulation in people with spinal cord injury: study protocol of a randomized clinical trial.

Background: The seemingly simple tasks of standing and walking require continuous integration of complex spinal reflex circuits between descending motor commands and ascending sensory inputs. Spinal cord injury greatly impairs standing and walking ability, but both improve with locomotor training. However, even after multiple locomotor training sessions, abnormal muscle activity and coordination persist. Thus, locomotor training alone cannot fully optimize the neuronal plasticity required to strengthen the synapses connecting the brain, spinal cord, and local circuits and potentiate neuronal activity based on need. Transcutaneous spinal cord (transspinal) stimulation alters motoneuron excitability over multiple segments by bringing motoneurons closer to threshold, a prerequisite for effectively promoting spinal locomotor network neuromodulation and strengthening neural connectivity of the injured human spinal cord. Importantly, whether concurrent treatment with transspinal stimulation and locomotor training maximizes motor recovery after spinal cord injury is unknown. Methods: Forty-five individuals with chronic spinal cord injury are receiving 40 sessions of robotic gait training primed with 30 Hz transspinal stimulation at the Thoracic 10 vertebral level. Participants are randomized to receive 30-minutes of active or sham transspinal stimulation during standing or active transspinal stimulation while supine followed by 30-minutes of robotic gait training. Over the course of locomotor training, the body weight support, treadmill speed, and leg guidance force are adjusted as needed for each participant based on absence of knee buckling during the stance phase and toe dragging during the swing phase. At baseline and after completion of all therapeutic sessions, neurophysiological recordings registering corticospinal and spinal neural excitability changes along with clinical assessment measures of standing and walking, and autonomic function via questionnaires regarding bowel, bladder and sexual function are taken. Discussion: The results of this mechanistic randomized clinical trial will demonstrate that tonic transspinal stimulation strengthens corticomotoneuronal connectivity and dynamic neuromodulation through posture-dependent corticospinal and spinal neuroplasticity. We anticipate that this mechanistic clinical trial will greatly impact clinical practice because in real-world clinical settings, noninvasive transspinal stimulation can be more easily and widely implemented than invasive epidural stimulation. Additionally, by applying multiple interventions to accelerate motor recovery, we are employing a treatment regimen that reflects a true clinical approach. Trial registration: ClinicalTrials.gov: NCT04807764; Registered on March 19, 2021.

Evaluation of efficacy of subgingival administration of 1% chlorhexidine gel as an adjunct to scaling and root planing in the treatment of chronic periodontitis - A clinical and microbiological study.

Aim: The aim of the present study was to evaluate the clinical and microbiological effects of subgingival administration of 1% chlorhexidine gel (Chlorhexamed® 1% gel) in patients with chronic periodontitis. Settings and Design: The study was done in a parallel-arm design with a total of 30 patients with 60 sites suffering from chronic periodontitis. The patients were divided into control and experimental groups. Materials and Methods: The clinical parameters recorded were plaque index, gingival index, modified sulcular bleeding index, probing pocket depth and relative attachment level at baseline, 1 month and 3 month. Microbiological colony-forming units were assessed for Porphyromonas gingivalis, Fusobacterium nucleatum and Tannerella forsythia at baseline, 1 week, 1 month and 3 months. The control group received scaling and root planing (SRP) after baseline evaluation; however, the experimental group received the application of Chlorhexamed® gel within 48 hours after SRP. Then, the values obtained were subjected to statistical analysis. Results: Both groups showed significant improvement from the baseline to 3 months in all clinical and microbiological parameters. The experimental group showed better improvement in all parameters. Conclusion: The use of Chlorhexamed® gel has proven to be an efficacious adjunct with SRP in the treatment of chronic periodontitis.

Adjuvants influence the maturation of VRC01-like antibodies during immunization.

Once naive B cells expressing germline VRC01-class B cell receptors become activated by germline-targeting immunogens, they enter germinal centers and undergo affinity maturation. Booster immunizations with heterologous Envs are required for the full maturation of VRC01-class antibodies. Here, we examined whether and how three adjuvants, Poly(I:C), GLA-LSQ, or Rehydragel, that activate different pathways of the innate immune system, influence the rate and type of somatic mutations accumulated by VRC01-class BCRs that become activated by the germline-targeting 426c.Mod.Core immunogen and the heterologous HxB2.WT.Core booster immunogen. We report that although the adjuvant used had no influence on the durability of plasma antibody responses after the prime, it influenced the plasma VRC01 antibody titers after the boost and the accumulation of somatic mutations on the elicited VRC01 antibodies.

Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy.

Analyses of HIV-1 envelope (Env) binding to CD4, and the conformational changes the interactions induce, inform the molecular mechanisms and factors governing HIV-1 infection. To address these questions, we used a single-molecule detection (SMD) approach to study the nature of reactions between soluble CD4 (sCD4) and soluble HIV-1 trimers. SMD of these reactions distinguished a mixture of one, two, or three CD4-bound trimer species. Single-ligand trimers were favored at early reaction times and ligand-saturated trimers later. Furthermore, some trimers occupied by one sCD4 molecule did not bind additional ligands, whereas the majority of two ligand-bound species rapidly transitioned to the saturated state. Quantification of liganded trimers observed in reactions with various sCD4 concentrations reflected an overall negative cooperativity in ligand binding. Collectively, our results highlight the general utility of SMD in studying protein interactions and provide critical insights on the nature of sCD4-HIV-1 Env interactions.

Overcoming Steric Restrictions of VRC01 HIV-1 Neutralizing Antibodies through Immunization.

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2∗02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

Prevalence of extended-spectrum beta-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital.

Extended-spectrum beta-lactamases (ESBLs) continue to be a major problem in clinical setups the world over, conferring resistance to the expanded-spectrum cephalosporins. Knowledge about their prevalence is essential to guide towards appropriate antibiotic treatment. The aim of the present study is to determine the prevalence of ESBL producers among Escherichia coli and Klebsiella pneumoniae isolates at a tertiary care institution. A total of 357 clinical isolates comprising E. coli (n = 181) and K. pneumoniae (n = 176) were recovered from various clinical samples over a period of six months from April to September 2006. Antibiogram profile of these isolates was determined to commonly used antibiotics, along with screening for ESBL production by the screening test as recommended by the Clinical Laboratory Standards Institute (CLSI). Isolates which showed positive results with screening test were shortlisted for confirmatory tests of ESBL production. Two tests were performed: phenotypic confirmatory test with combination disk and the minimum inhibitory concentration (MIC) reduction test. Out of 357 isolates of E. coli and K. pneumoniae screened for ESBL production, 120 were found to be potential ESBL producers. Of these, 80 isolates were confirmed to be ESBL producers. Thus the prevalence of ESBL-producing isolates of E. coli and K. pneumoniae was found to be 22% (80 out of 357). This was significantly lower than the data available from other hospitals.

Drosophila CRY Entrains Clocks in Body Tissues to Light and Maintains Passive Membrane Properties in a Non-clock Body Tissue Independent of Light.

Circadian (∼24 hr) clocks regulate daily rhythms in physiology, metabolism, and behavior via cell-autonomous transcriptional feedback loops. In Drosophila, the blue-light photoreceptor CRYPTOCHROME (CRY) synchronizes these feedback loops to light:dark cycles by binding to and degrading TIMELESS (TIM) protein. CRY also acts independently of TIM in Drosophila to alter potassium channel conductance in arousal neurons after light exposure, and in many animals CRY acts independently of light to repress rhythmic transcription. CRY expression has been characterized in the Drosophila brain and eyes, but not in peripheral clock and non-clock tissues in the body. To investigate CRY expression and function in body tissues, we generated a GFP-tagged-cry transgene that rescues light-induced behavioral phase resetting in cry03 mutant flies and sensitively reports GFP-CRY expression. In bodies, CRY is detected in clock-containing tissues including Malpighian tubules, where it mediates both light-dependent TIM degradation and clock function. In larval salivary glands, which lack clock function but are amenable to electrophysiological recording, CRY prevents membrane input resistance from falling to low levels in a light-independent manner. The ability of CRY to maintain high input resistance in these non-excitable cells also requires the K+ channel subunits Hyperkinetic, Shaker, and ether-a-go-go. These findings for the first time define CRY expression in Drosophila peripheral tissues and reveal that CRY acts together with K+ channels to maintain passive membrane properties in a non-clock-containing peripheral tissue independent of light.

An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

Comparative clinical evaluation of glycosylated haemoglobin level in healthy and chronic periodontitis patients: A chairside diagnostic method.

OBJECTIVE AND BACKGROUND: Glycosylated haemoglobin (HbA1c) level can consequently be interpreted as an average of the blood glucose present over the past 3-4 months. Periodontitis is associated with glycemic control in patients with diabetes. The purpose of this study was to determine the level of HbA1c in healthy and periodontitis patients who were previously not diagnosed with diabetes mellitus. MATERIALS AND METHODS: A total of 40 patients were selected for study and divided into two groups. Group 1 included patients with a healthy periodontium, and Group 2 included patients suffering from chronic periodontitis. Finger stick blood was collected by special collection unit (A1CNOW+® Bayer Health Care, Tarrytown New York, USA), for estimating level of HbA1c. RESULT: Both groups showed similar HbA1c levels clinically with slight increase in levels in the test group, but was statistically significant (test--5.66 ± 0.35%, control--5.17 ± 0.3% P = 0.003). CONCLUSION: Indians are at a high-risk of developing periodontitis and diabetes. These data suggest a possible link between periodontitis and glycemic control in nondiabetic individuals, periodontal disease may be a potential contributor to the development of type 2 diabetes.

Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi.

AIM: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. MATERIALS AND METHODS: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. RESULTS: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 microg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 microg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83-->Tyr or Ser 83-->Phe), whereas double mutations (Ser 83-->Phe and Asp 87-->Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation. CONCLUSION: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.