Rational protein engineering to enhance MHC-independent T cell receptors.

Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T cell response, we designed MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant chains. Using predictive modeling, we observed that this standard "cut and paste" approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable-constant domain interface. Through iterative modeling and sequence modifications we developed structure-enhanced miTCRs which significantly improved receptor-driven T cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T cell persistence and enabled improved leukemic control in vivo compared to classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function.

Transcriptional analysis of primary ciliary dyskinesia airway cells reveals a dedicated cilia glutathione pathway.

Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA sequencing, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, healthy individuals, and induced pluripotent stem (iPS) cells generated from a PCD patient. Transcriptomic analysis revealed unique signatures in PCD airway cells compared to their mothers and healthy individuals. Gene expression in heterozygous mothers' cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases, GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism, Chlamydomonas reinhardtii, demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

To Evaluate the Effect of Vitamin B Complex on Wound Healing - A Clinical and Microbiological Study.

Background: The purpose of this study is to explore the effect of vitamin B complex supplementation following periodontal flap surgery on clinical and microbiological parameters. Materials and Methods: A randomized controlled trial on 10 patients with periodontitis in split-mouth design was undertaken to find the effect of vitamin B complex supplementation with open flap debridement on periodontal wound healing. Multiplex polymerase chain reaction (PCR) for Tannerella forsythus and Porphyromonas gingivalis was done using subgingival plaque samples at 0 and 90th day. Results: The results showed a significant reduction (P < 0.01) of clinical (plaque index, gingival index, gingival bleeding index, probing pocket depth, and relative attachment level) and microbial profile in both treatment groups, whereas on intergroup analysis, more reduction in all clinical parameters were observed in the test group, but statistically, the results were insignificant.

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5 . Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief Summary: A mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

Contribution of integrin adhesion to cytokinetic abscission and genomic integrity.

Recent research shows that integrin-mediated adhesion contributes to the regulation of cell division at two key steps: the formation of the mitotic spindle at the mitotic entry and the final cytokinetic abscission at the mitotic exit. Failure in either of these processes will have a direct impact on the other in each round of the cell cycle and on the genomic integrity. This review aims to present how integrin signals are involved at these cell cycle stages under normal conditions and some safety mechanisms that may counteract the generation of aneuploid cells in cases of defective integrin signals.

Integrin-Mediated Adhesion Promotes Centrosome Separation in Early Mitosis.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

The TERB1 MYB domain suppresses telomere erosion in meiotic prophase I.

The meiosis-specific telomere-binding protein TERB1 anchors telomeres to the nuclear envelope and drives chromosome movements for the pairing of homologous chromosomes. TERB1 has an MYB-like DNA-binding (MYB) domain, which is a hallmark of telomeric DNA-binding proteins. Here, we demonstrate that the TERB1 MYB domain has lost its canonical DNA-binding activity. The analysis of Terb1 point mutant mice expressing TERB1 lacking its MYB domain showed that the MYB domain is dispensable for telomere localization of TERB1 and the downstream TERB2-MAJIN complex, the promotion of homologous pairing, and even fertility. Instead, the TERB1 MYB domain regulates the enrichment of cohesin and promotes the remodeling of axial elements in the early-to-late pachytene transition, which suppresses telomere erosion. Considering its conservation across metazoan phyla, the TERB1 MYB domain is likely to be important for the maintenance of telomeric DNA and thus for genomic integrity by suppressing meiotic telomere erosion over long evolutionary timescales.

Abortion quality of care from the client perspective: a qualitative study in India and Kenya.

Quality healthcare is a key part of people's right to health and dignity, yet access to high-quality care can be limited by legal, social and economic contexts. There is limited consensus on what domains constitute quality in abortion care and the opinions of people seeking abortion have little representation in current abortion quality measures. In this qualitative study, we conducted 45 interviews with abortion clients in Mumbai, India, and in Eldoret and Thika, Kenya, to assess experiences with abortion care, definitions of quality and priorities for high-quality abortion care. Among the many aspects of care that mattered to clients, the client-provider relationships emerged as essential. Clients prioritized being treated with kindness, respect and dignity; receiving information and counselling that was personalized to their individual situation and reassurance and support from their provider throughout the entire abortion process, including follow-up after the abortion. Many clients also noted the importance of skilled providers and appropriate care. There were similarities across the two country contexts, yet there were some differences in how clients defined high-quality care; therefore, specific political and cultural influences must be considered when implementing measurement and improving person-centred quality of care. These domains, particularly interpersonal interactions, should be prioritized in India and Kenya when health systems, facilities and providers design person-centred measures for quality in abortion care.

Structural equation modeling for factors influencing patients' willingness to replace removable dentures.

PURPOSE: To investigate the factors influencing patients' willingness to replace removable dentures using structural equation modeling (SEM). METHODS: A total of 153 patients who sought consultations for removable dentures self-evaluated denture quality using a visual analog scale (VAS); health-related quality of life using the 36-item Short-Form Health Survey; and oral health-related quality of life using the Oral Health Impact Profile. Dental clinicians evaluated denture quality using a VAS and by assessing the presence of defects. After being informed of various treatments, patients were asked whether they would prefer denture replacement. SEM was applied to analyze the relationships and interactive effects among the variables. RESULTS: The final model showed high goodness-of-fit indices (chi-square/degree of freedom = 1.009, comparative fit index = 1.000, Tucker-Lewis index = 0.999, standardized root mean square residual = 0.421, and root mean square error of approximation = 0.008). SEM demonstrated that two latent constructs indirectly predicted patients' willingness to replace dentures; the standardized total effects of good oral health and poor denture quality were -0.154 and 0.503, respectively. CONCLUSION: These findings provide a unified understanding of the shared decision-making process for denture replacement and highlight the pretreatment assessments that play a relevant role in patient treatment preferences.

Exploring stigma and social norms in women's abortion experiences and their expectations of care.

Abortion is a common and essential component of sexual and reproductive health care, yet social norms and stigma influence women's decision-making and create barriers to safe abortion care. This qualitative study in Kenya and India explores abortion-related fears, expectations and perceptions of stigma among women who have obtained abortion services. In 2017, we conducted 34 semi-structured interviews and 2 focus groups with women who had obtained abortion services in Maharashtra state in India and Thika and Eldoret in Kenya. Thematic analysis was informed by the individual-level abortion stigma framework and theory of normative conduct. We aimed to learn about the diversity of women's experiences, analysing pooled data from the two countries. Most participants reported that before seeking abortion they had little prior knowledge about the service, expected to be judged during care, and feared the service would be ineffective or have negative health consequences. Many reported that community members disapprove of abortion and that a woman's age or marital status could exacerbate judgement. Some reported limiting disclosure of their abortion to avoid judgement. Negative stories, the secrecy around abortion, perceived stigma, social norms, and fear of sanctions all contributed to women's fears and low expectations. These findings elucidate the relationship between social norms and stigma and how expectations and concerns affect women's experiences seeking care. The results have implications for practice, with potential to inform improvements to services and help organisations address stigma as a barrier to care. This may be particularly relevant for younger or unmarried women.

Septin and Ras regulate cytokinetic abscission in detached cells.

BACKGROUND: Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) ± overactive HRas. RESULTS: In contrast to BJ and BJ-LT cells, non-adherent BJ-LT-Ras cells recruited ALIX and CHMP4B to the midbody and divided. In detached BJ and BJ-LT cells regression of the cytokinetic furrow was suppressed by intercellular bridge-associated septin; after re-adhesion these cells divided by cytofission, however, some cells became bi-nucleated because of septin reorganization and furrow regression. Adherent bi-nucleated BJ cells became senescent in G1 with p21 accumulation in the nucleus, apparently due to p53 activation since adherent bi-nucleated BJ-LT cells passed through next cell cycle and divided into mono-nucleated tetraploids; the two centrosomes present in bi-nucleated BJ cells fused after furrow regression, pointing to the PIDDosome pathway as a possible mechanism for the p53 activation. CONCLUSIONS: Several mechanisms prevent detached normal cells from generating tumor-causing tetraploid cells unless they have a suppressed p53 response by viruses, mutation or inflammation. Importantly, activating Ras mutations promote colony growth of detached transformed cells by inducing anchorage-independent cytokinetic abscission in single cells.

Distinct histone modifications denote early stress-induced drug tolerance in cancer.

Besides somatic mutations or drug efflux, epigenetic reprogramming can lead to acquired drug resistance. We recently have identified early stress-induced multi-drug tolerant cancer cells termed induced drug-tolerant cells (IDTCs). Here, IDTCs were generated using different types of cancer cell lines; melanoma, lung, breast and colon cancer. A common loss of the H3K4me3 and H3K27me3 and gain of H3K9me3 mark was observed as a significant response to drug exposure or nutrient starvation in IDTCs. These epigenetic changes were reversible upon drug holidays. Microarray, qRT-PCR and protein expression data confirmed the up-regulation of histone methyltransferases (SETDB1 and SETDB2) which contribute to the accumulation of H3K9me3 concomitantly in the different cancer types. Genome-wide studies suggest that transcriptional repression of genes is due to concordant loss of H3K4me3 and regional increment of H3K9me3. Conversely, genome-wide CpG site-specific DNA methylation showed no common changes at the IDTC state. This suggests that distinct histone methylation patterns rather than DNA methylation are driving the transition from parental to IDTCs. In addition, silencing of SETDB1/2 reversed multi drug tolerance. Alterations of histone marks in early multi-drug tolerance with an increment in H3K9me3 and loss of H3K4me3/H3K27me3 is neither exclusive for any particular stress response nor cancer type specific but rather a generic response.

Tension-induced cytokinetic abscission in human fibroblasts.

Previous studies have shown that cytokinetic abscission at the end of mitosis is executed by the ESCRT machinery in mammalian cells, and that the process is dependent on adhesion-induced integrin signalling via a FAK-PLK1-CEP55-TSG101/Alix-CHMP4B pathway. The present study identified an alternative abscission mechanism driven by mechanical force. In the absence of integrin signals (non-adherent conditions), cytokinesis in non-transformed human fibroblasts proceeds to CEP55 accumulation at the midbody, but after prolonged time (>3 hours) the major midbody components Aurora B, MKLP1 and CEP55 were no longer detected in the area. Upon adhesion to fibronectin, such cells were able to complete abscission without re-appearance of midbody proteins. Live-cell imaging revealed that re-plating on stiff fibronectin matrix (64 KPa) allowed >95% of the cells to complete abscission within 9 hours while the corresponding number was 40% on soft fibronectin matrix (0.5 KPa). The cells re-plated on poly-L-lysine were not able to generate tension and did not divide. Thus, mechanical tension can cause cytokinetic abscission by stretching of the intercellular bridge between the two daughter cells until it eventually ruptures without the involvement of ESCRT complexes. Importantly, regression of the cleavage furrow and formation of bi-nucleated cells did not occur in most of the suspension-treated mitotic cells after re-plating on fibronectin. Septin, which stabilizes the membrane associated with the midbody, was found to remain along the ingressed membrane, suggesting that this filament system maintains the membrane bridge although the midbody had dissolved, thereby preventing regression and allowing tension to act on the narrow intercellular bridge.

Comparative Study of Oral Gabapentin, Pregabalin, and Clonidine as Premedication for Anxiolysis, Sedation, and Attenuation of Pressor Response to Endotracheal Intubation.

INTRODUCTION: The aim of the present study was to evaluate and compare the effect of clonidine 200 µg and gabapentin 900 mg and pregabalin 150 mg in attenuation of the hemodynamic response to laryngoscopy and intubation in normotensive patients undergoing elective surgery. METHODS: Ninety adult patients between 18 and 60 years are enrolled in the study. Patients with American Society of Anesthesiologists Grade-I and Grade-II are included which are posted for elective surgery under general anesthesia. Patients were divided into three groups: A, B, and C and received oral drugs 90 min before induction of general anesthesia, pregabalin 150, gabapentin 900mg, and clonidine 200 µg, respectively. Hemodynamic parameters such as heart rate and blood pressure were noted just before the (basal) administration of the drug, and in operation room, readings were recorded before intubation (T0) and after intubation at 1, 3, 5, and 10 min. Sedation and anxiety score were noted after 1 h of oral administration of the drug. RESULTS: Mean arterial pressure was well attenuated by pregabalin than others, and mean heart rate following laryngoscopy and intubation was attenuated by clonidine group significantly. CONCLUSION: We conclude that oral pregabalin and gabapentin attenuate blood pressure response fairly well and heart rate significantly attenuated by clonidine. All three drugs are very effective for relieving anxiety and improving sedation.

Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody.

Adhesion to extracellular matrix is required for cell cycle progression through the G1 phase and for the completion of cytokinesis in normal adherent cells. Cancer cells acquire the ability to proliferate anchorage-independently, a characteristic feature of malignantly transformed cells. However, the molecular mechanisms underlying this escape of the normal control mechanisms remain unclear. The current study aimed to identify adhesion-induced reactions regulating the cytokinesis of non-transformed human fibroblasts.The adhesion-dependent control of cytokinesis was found to occur at a late stage close to the abscission, during which the endosomal sorting complex required for transport (ESCRT) severs the thin intercellular bridge connecting two nascent daughter cells. CEP55, a key protein involved in the abscission process, was localized at the midbody in both adherent and non-adherent fibroblasts, but it was unable to efficiently recruit ALIX, TSG101, and consequently the ESCRT-III subunit CHMP4B was missing in the non-adherent cells. PLK1, a kinase that prevents premature recruitment of CEP55 to the midbody, disappeared from this site more rapidly in the non-adherent cells. A FAK-Src signaling pathway downstream of integrin-mediated cell adhesion was found to decelerate both PLK1 degradation and CEP55 accumulation at the midbody. These data identify the regulation of PLK1 and CEP55 as steps where integrins exert control over the cytokinetic abscission.

Nanocurcumin: a promising therapeutic advancement over native curcumin.

Curcumin, a naturally occurring polyphenolic compound, is known to have a wide range of therapeutic and pharmacological properties. Although it is a considerably promising compound, its poor water solubility and fast degradation profile make it compromise over its bioavailability way below the threshold level on administration. Over a period of time, a lot of emphasis has been given to improve the biodistribution of native curcumin, but it is only recently that the application of the field of nanotherapeutics has significantly improved its therapeutic efficacy. This is through the development of nanorange formulations of curcumin, popularly known as the "nanocurcumin." These attempts have given a strong platform to reap all the biological benefits from this phytodrug, which was not significantly plausible earlier. This review gives an insight into the reasons that make nanocurcumin a more therapeutically advanced drug than its native counterpart. It also discusses various nanometric formulations of curcumin that have been reported for its controlled and targeted delivery along with a critical comparison of its therapeutic efficacy with free curcumin. We also summarize the biological applications, patented technologies, and current status of the ongoing clinical trials related to nanocurcumin.

Preventive efficacy of bulk and nanocurcumin against lead-induced oxidative stress in mice.

Chronic lead exposure is associated with several health disorders in humans and animals. Lead exposure leads to the generation of reactive oxygen species and depletes body antioxidant enzymes causing damage to various macromolecules and ultimately cell death. Curcumin has been widely recognized to protect against metal toxicity but has major limitations of reduced bioavailability. Nanoencapsulation of curcumin could be an effective strategy to combat lead induced toxic manifestations. The present study investigates the protective efficacy of bulk and nanocurcumin against lead-induced toxicity. Swiss albino mice were daily exposed to lead acetate (25 mg/kg, i.p.) alone and after 1 h treated either with curcumin (15 mg/kg, orally) or nanocurcumin (15 mg/kg, orally) for two consecutive weeks. The preventive efficacy of nanocurcumin was evaluated against various altered biochemical variables suggestive of oxidative stress and lead accumulation in blood and soft tissues. Coadministration of nanocurcumin with lead restored the altered δ-aminolevulinic acid dehydratase activity, glutathione (reduced and oxidized) levels, and also decreased reactive oxygen species, and thiobarbituric acid reactive substances levels. Nanocurcumin due to its possible chelating property and enhanced bioavailability efficiently removed lead from blood and soft tissues compared to bulk curcumin. Results demonstrate the enhanced preventive efficacy of nanocurcumin and suggest an interesting and novel approach for better treatment of lead toxicity.

Dental implant survival in diabetic patients; review and recommendations.

Rising population of diabetic individuals across the world has become a big concern to the society. The persistent hyperglycemia may affect each and every tissue and consequently results in morbidity and eventually mortality in diabetic patients. A direct negative response of diabetes has been observed on oral tissues with few contradictions however, little are known about effect of diabetes on dental implant treatment and the consequent results. Many studies concerned with osteointegration and prognosis of dental implant in diabetic patients have been conducted and published since 1994. These studies have been critically reviewed to understand the impact of diabetes on the success of dental implant and the factors to improve osseointegration and consequently survival of dental implant in diabetic patients. Theoretical literatures and studies in diabetic animals substantiate high failure rate of implants but most of clinical studies indicated statistically insignificant failure of dental implants even in moderately uncontrolled diabetic patients. Success of dental implant in well and fairly controlled diabetic patients with proper treatment planning, prophylactic remedies and adequate postsurgical maintenance appears as good as normal individuals.