Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Mediator complex: an important regulator of root system architecture.

Mediator, a multiprotein complex, is an important component of the transcription machinery. In plants, the latest reports from our group and some other studies have established that Mediator functions as a signal processor that conveys transcriptional signals from transcription factors to RNA polymerase II. It has been found to be involved in different developmental and stress-adaptation conditions ranging from embryo, root, and shoot development to flowering and senescence and also in response to different biotic and abiotic stresses. In the last one decade, significant progress has been made in understanding the role of Mediator subunits in root development. They have been shown to transcriptionally regulate development of almost all the components of root system architecture - primary root, lateral root and root hair. Their role has also been appreciated in nutrient acquisition through root. In this review, we have discussed all the known functions of Mediator subunits during root development. We have also highlighted the role of Mediator as a nodal point for processing different hormone signaling that regulate root morphogenesis and growth.

A data decomposition-based hierarchical classification method for multi-label classification of contractual obligations for the purpose of their governance.

Contract governance ensures that the agreed outcomes between customers and vendors are fulfilled. Information Technology (IT) outsourcing organizations enter thousands of contractual relationships each month leading to a high volume of business-critical contractual text that must be reviewed and deciphered for effective governance. The key to effective governance of contracts is a model that facilitates assigning ownership of the obligations to the right departments in an organization and allocating their accountability to the right stakeholders. For this, the contractual obligations need to be identified and classified so that details such as actions to be taken by departments in an organization and their ownership as per a given clause are brought out for the purpose of governance. In this paper, we present our work on automated extraction and classification of obligations present in Software Engineering (SE) contracts for the purpose of contracts governance. We propose a novel data decomposition-based hierarchical classification method for a multi-label classification of contractual obligations. We conducted experiments for a fine-grained automated classification of more than 55,000 statements from 50 large real-life SE contract documents received from a large vendor organization into 152 governance-specific classes. The results indicate that the proposed method can bring about a 7-8% improvement in accuracies when compared to state-of-the-art classification baselines such as BERT, RoBERTa, and generative models such as GPT-2.

Synthesis of 2,2-dimethyl-chroman-based stereochemically flexible and constrained anti-breast cancer agents.

Receptors are proteinous macromolecules which remain in the apo form under normal/unliganded conditions. As the ligand approaches, there are specific stereo-chemical changes in the apo form of the receptor as per the stereochemistry of a ligand. Accordingly, a series of substituted dimethyl-chroman-based stereochemically flexible and constrained Tamoxifen analogs were synthesized as anti-breast cancer agents. The synthesized compounds 19a-e, 20a-e, 21, and 22a-e, showed significant antiproliferative activity against estrogen receptor-positive (ER+, MCF-7) and negative (ER-, MDA MB-231) cells within IC50 value 8.5-25.0 µM. Amongst all, four potential molecules viz 19b, 19e, 22a, and 22c, were evaluated for their effect on the cell division cycle and apoptosis of ER+ and ER- cancer cells (MCF-7 & MDA MB-231cells), which showed that these compounds possessed antiproliferative activity through triggering apoptosis. In-silico docking experiments elucidated the possible affinity of compounds with estrogen receptors-α and -ß.

Self-assembled monolayer of 2-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)phenol (MTP) on gold surface: A platform for impedimetric and potentiometric sensing of mercury(II).

2-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)phenol (MTP) was synthesized, self-assembled on the surface of gold (Au) electrode (Au-MTP) followed by characterization using Cyclic voltammetry (CV) and Electrochemical impedance spectroscopy (EIS). CV and EIS confirmed the formation of well-organized Au-MTP SAM free from defects and pinholes. Au-MTP was further utilized as a platform for sensing of Hg2+ using EIS. The results showed sensitive and selective response of Au-MTP towards Hg2+ in the linear concentration range from 1.0 × 10-10 M to 1.0 × 10-4 M with limit of detection (LoD) of 5.6 × 10-11 M. Furthermore, MTP was self-assembled on gold nanoparticles (AuNPs) and MTP bound gold nanoparticles (MTP-AuNPs) so obtained were used as modifier for construction of carbon paste electrode (CPE). Hg2+-CPE exhibited Nernstian response towards Hg2+ with slope of 28.3 mV/decade in the concentration range from 1.0 × 10-5 M to 1.0 × 10-1 M with LoD of 6.3 × 10-6 M. Both the Au-MTP EIS sensor and Hg2+-CPE were successfully applied for estimation of Hg2+ content in tap water samples.

Dynamic interplay of microtubule and actomyosin forces drive tissue extension.

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

Effect of Position of Donor Units and Alkyl Groups on Dye-Sensitized Solar Cell Device Performance: Indoline-Aniline Donor-Based Visible Light Active Unsymmetrical Squaraine Dyes.

Indoline (In) and aniline (An) donor-based visible light active unsymmetrical squaraine (SQ) dyes were synthesized for dye-sensitized solar cells (DSSCs), where the position of An and In units was changed with respect to the anchoring group (carboxylic acid) to have In-SQ-An-CO2H and An-SQ-In-CO2H sensitizers, AS1-AS5. Linear or branched alkyl groups were functionalized with the N atom of either In or An units to control the aggregation of the dyes on TiO2. AS1-AS5 exhibit an isomeric π-framework where the squaric acid unit is placed in the middle, where AS2 and AS5 dyes possess the anchoring group connected with the An donor, and AS1, AS3, and AS4 dyes having the anchoring group connected with the In donor. Hence, the conjugation between the middle squaric acid acceptor unit and the anchoring -CO2H group is short for AS2, AS5, and AK2 and longer for AS1, AS3, and AS4 dyes. AS dyes showed absorption between 501 and 535 nm with extinction coefficients of 1.46-1.61 × 105 M-1 cm-1. Further, the isomeric π-framework of An-SQ-In-CO2H and In-SQ-An-CO2H exhibited by means of changing the position of In and An units a bathochromic shift in the absorption properties of AS2 and AS5 compared to the AS1, AS3, and AS4 dyes. The DSSC device fabricated with the dyes contains short acceptor-anchoring group distance (AS2 and AS5) showed high photovoltaic performances compared to the dyes having longer distance (AS1, AS3, and AS4) with the iodolyte (I-/I3-) electrolyte. DSSC device efficiencies of 5.49, 6.34, 6.16, and 5.57% have been achieved for AS1, AS2, AS3, and AS4 dyes, respectively; without chenodeoxycholic acid (CDCA), small changes have been observed in the device performance of the AS dyes with CDCA. Significant changes have been noted in the DSSC parameters (open-circuit voltage VOC, short-circuit current JSC, fill factor ff, and efficiency η) for the AS5 dye while sensitized with CDCA and showed highest DSSC efficiency of 8.01% in the AS dye series. This study revealed the potential of shorter SQ acceptor-anchoring group distance over the longer one and the importance of alkyl groups on the overall DSSC device performance for the unsymmetrical squaraine dyes.

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

A scalable and cost-efficient rRNA depletion approach to enrich RNAs for molecular biology investigations.

Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A)- RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed rRNA Removal by RNaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.

Intermittent Fasting and Vitamin E Supplementation Attenuates Hypothyroidism-Associated Ophthalmopathy.

Visualization is a complex-integrated procedure of the eyes and brain that allows to see this colorful world. Hypothyroidism-associated ophthalmopathy (HAO), often known as dry eyes, swelling around the eyes, blurred vision, glaucoma, and cataracts, are some eye-related issues caused by hypothyroidism. Yet there is no permanent cure for hypothyroidism; taking medicine throughout life is the only solution to keep its harmful effects under control. This study used intermittent fasting (IF) and vitamin E (Vit.E) supplementation to prevent hypothyroidism-associated ophthalmopathy. This study hypothesized that intermittent fasting-like diet regimens and vitamin supplementation should reduce the propagation of HAO by its antioxidant potential. In the present study, experimental animals are divided into five groups: normal, hypothyroidism control, dual, Vit. E, and IF. Hypothyroidism is generated in the experimental groups by taking propylthiouracil (PTU) for 24 days while also taking IF and Vit. E supplements. The hypothyroid-induced experimental animals demonstrated an increase in IOP and lipid peroxidation while thyroid hormone levels depicted a massive decline which is a clear denotation of the effects of the thyroid on eyes and lifestyle. Ancient Ayurveda inspires these proposed therapies and has successfully reduced all the damage to the thyroid gland and the eye.

A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

Exploring multisite heterogeneity of human basal cell carcinoma proteome and transcriptome.

Basal cell carcinoma (BCC) is the most common type of skin cancer. Due to multiple, potential underlying molecular tumor aberrations, clinical treatment protocols are not well-defined. This study presents multisite molecular heterogeneity profiles of human BCC based on RNA and proteome profiling. Three areas from lesions excised from 9 patients were analyzed. The focus was gene expression profiles based on proteome and RNA measurements of intra-tumor heterogeneity from the same patient and inter-tumor heterogeneity in nodular, infiltrative, and superficial BCC tumor subtypes from different patients. We observed significant overlap in intra- and inter-tumor variability of proteome and RNA expression profiles, showing significant multisite heterogeneity of protein expression in the BCC tumors. Inter-subtype analysis has also identified unique proteins for each BCC subtype. This profiling leads to a deeper understanding of BCC molecular heterogeneity and potentially contributes to developing new sampling tools for personalized diagnostics therapeutic approaches to BCC.

Ligand-mediated Targeted Drug Delivery Approaches against Hepatocellular Carcinoma.

One of the most important health problems in the world today is cancer. The World Health Organization (WHO) reported that it results in 8.9 million deaths annually. Malignant tumours and unregulated cell proliferation are features of malignant neoplasms, which can also invade nearby body regions. Hepatocellular carcinoma is the third most prevalent cause of cancer-related death worldwide and the fifth most common kind of cancer, according to a recent analysis. Patients with liver disease as well as chronic hepatitis B and C are more likely to develop hepatocellular carcinoma (HCC). Physical barriers, including RES absorption, opsonization, and first-pass drug metabolism, make drug therapy more challenging. Conventional cancer therapy procedures have a low response rate or may continue to be unsuccessful due to multi-drug resistance (MDR), high clearance rates, and other side effects because of suboptimal drug distribution and insufficient drug concentration reaching cancer cells. Innovative target drug molecules that are tailored to the injured liver cells must be developed in order to improve medication administration and drug targeting. The use of targeting ligands that have been joined to drug molecules or nanocarriers forms the basis of innovative targeting techniques. After being conjugated with the treatment method, ligands for targeting hepatocellular carcinoma cells included asialoglycoprotein, galactoside, lactobionic acid, mannose-6-phosphate, PDGF, antibodies, and aptamers.

Machine learning-based ensemble approach in prediction of lung cancer predisposition using XRCC1 gene polymorphism.

The employment of machine learning approaches has shown promising results in predicting cancer. In the current study, polymorphisms data of five single nucleotide polymorphisms (SNPs) of DNA repair gene XRCC1 (XRCC1 399, XRCC1 194, XRCC1 206, XRCC1 632, XRCC1 280) of the north Indian population along with four smoking status data is considered as an input to the proposed ensemble model to predict the risk of individual susceptibility to the lung cancer. The prediction accuracy of the proposed ensemble model for cancer predisposition was found to be 85%. The model performance is also evaluated using sensitivity, specificity, precision and the Gini index, which is found in the range of 0.83-0.87. The proposed model also outperformed in all evaluation parameters when compared with the individual Model (LM, SVM, RF, KNN and baseline neural net). Collectively, current results suggest the potential of the proposed ensemble model in predicting the risk of cancer based on XRCC1 SNPs data.Communicated by Ramaswamy H. Sarma.

Role of GLP-1 receptor agonist in diabetic cardio-renal disorder: Recent updates of clinical and pre-clinical evidence.

Cardiovascular complications and renal disease is the growing cause of mortality in patients with diabetes. The subversive complications of diabetes such as hyperglycemia, hyperlipidemia and insulin resistance lead to an increase in the risk of myocardial infarction (MI), stroke, heart failure (HF) as well as chronic kidney disease (CKD). Among the commercially available anti-hyperglycemic agents, incretin-based medications appear to be safe and effective in the treatment of type 2 diabetes mellitus (T2DM) and associated cardiovascular and renal disease. Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have been shown to be fruitful in reducing HbA1c, blood glucose, lipid profile, and body weight in diabetic patients. Several preclinical and clinical studies revealed the safety, efficacy, and preventive advantages of GLP-1RAs against diabetes-induced cardiovascular and kidney disease. Data from cardio-renal outcome trials had highlighted that GLP-1RAs protected people with established CKD from significant cardiovascular disease, lowered the likelihood of hospitalization for heart failure (HHF), and lowered all-cause mortality. They also had a positive effect on people with end-stage renal disease (ESRD) and CKD. Beside clinical outcomes, GLP-1RAs reduced oxidative stress, inflammation, fibrosis, and improved lipid profile pre-clinically in diabetic models of cardiomyopathy and nephropathy that demonstrated the cardio-protective and reno-protective effect of GLP-1RAs. In this review, we have focused on the recent clinical and preclinical outcomes of GLP-1RAs as cardio-protective and reno-protective agents as GLP-1RAs medications have been demonstrated to be more effective in treating T2DM and diabetes-induced cardiovascular and renal disease than currently available treatments in clinics, without inducing hypoglycemia or weight gain.

Understanding the role of hyperglycemia and the molecular mechanism associated with diabetic neuropathy and possible therapeutic strategies.

Diabetic neuropathy is a neuro-degenerative disorder that encompasses numerous factors that impact peripheral nerves in the context of diabetes mellitus (DM). Diabetic peripheral neuropathy (DPN) is very prevalent and impacts 50% of diabetic patients. DPN is a length-dependent peripheral nerve lesion that primarily causes distal sensory loss, discomfort, and foot ulceration that may lead to amputation. The pathophysiology is yet to be fully understood, but current literature on the pathophysiology of DPN revolves around understanding various signaling cascades involving the polyol, hexosamine, protein-kinase C, AGE, oxidative stress, and poly (ADP ribose) polymerase pathways. The results of research have suggested that hyperglycemia target Schwann cells and in severe cases, demyelination resulting in central and peripheral sensitization is evident in diabetic patients. Various diagnostic approaches are available, but detection at an early stage remains a challenge. Traditional analgesics and opioids that can be used "as required" have not been the mainstay of treatment thus far. Instead, anticonvulsants and antidepressants that must be taken routinely over time have been the most common treatments. For now, prolonging life and preserving the quality of life are the ultimate goals of diabetes treatment. Furthermore, the rising prevalence of DPN has substantial consequences for occupational therapy because such therapy is necessary for supporting wellness, warding off other chronic-diseases, and avoiding the development of a disability; this is accomplished by engaging in fulfilling activities like yoga, meditation, and physical exercise. Therefore, occupational therapy, along with palliative therapy, may prove to be crucial in halting the onset of neuropathic-symptoms and in lessening those symptoms once they have occurred.

Cell cycle dynamics control fluidity of the developing mouse neuroepithelium.

As developing tissues grow in size and undergo morphogenetic changes, their material properties may be altered. Such changes result from tension dynamics at cell contacts or cellular jamming. Yet, in many cases, the cellular mechanisms controlling the physical state of growing tissues are unclear. We found that at early developmental stages, the epithelium in the developing mouse spinal cord maintains both high junctional tension and high fluidity. This is achieved via a mechanism in which interkinetic nuclear movements generate cell area dynamics that drive extensive cell rearrangements. Over time, the cell proliferation rate declines, effectively solidifying the tissue. Thus, unlike well-studied jamming transitions, the solidification uncovered here resembles a glass transition that depends on the dynamical stresses generated by proliferation and differentiation. Our finding that the fluidity of developing epithelia is linked to interkinetic nuclear movements and the dynamics of growth is likely to be relevant to multiple developing tissues.

Mycobacterium tuberculosis PptT Inhibitors Based on Heterocyclic Replacements of Amidinoureas.

4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme for Mycobacterium tuberculosis (Mtb) survival and virulence and therefore an attractive target for a tuberculosis therapeutic. In this work, two modeling-informed approaches toward the isosteric replacement of the amidinourea moiety present in the previously reported PptT inhibitor AU 8918 are reported. Although a designed 3,5-diamino imidazole unexpectedly adopted an undesired tautomeric form and was inactive, replacement of the amidinourea moiety afforded a series of active PptT inhibitors containing 2,6-diaminopyridine scaffolds.

TLR4 activation by lysozyme induces pain without inflammation.

Mostly, pain has been studied in association with inflammation, until recent studies which indicate that during bacterial infections, pain mechanisms could be independent of the inflammation. Chronic pain can sustain long after the healing from the injury, even in the absence of any visible inflammation. However, the mechanism behind this is not known. We tested inflammation in lysozyme-injected mice foot paw. Interestingly, we observed no inflammation in mice foot paw. Yet, lysozyme injections induced pain in these mice. Lysozyme induces pain in a TLR4-dependent manner and TLR4 activation by its ligands such as LPS leads to inflammatory response. We compared the intracellular signaling of MyD88 and TRIF pathways upon TLR4 activation by lysozyme and LPS to understand the underlying mechanism behind the absence of an inflammatory response upon lysozyme treatment. We observed a TLR4 induced selective TRIF and not MyD88 pathway activation upon lysozyme treatment. This is unlike any other previously known endogenous TLR4 activators. A selective activation of TRIF pathway by lysozyme induces weak inflammatory cytokine response devoid of inflammation. However, lysozyme activates glutamate oxaloacetate transaminase-2 (GOT2) in neurons in a TRIF-dependent manner, resulting in enhanced glutamate response. We propose that this enhanced glutaminergic response could lead to neuronal activation resulting in pain sensation upon lysozyme injections. Collectively we identify that TLR4 activation by lysozyme can induce pain in absence of a significant inflammation. Also, unlike other known TLR4 endogenous activators, lysozyme does not activate MyD88 signaling. These findings uncover a mechanism of selective activation of TRIF pathway by TLR4. This selective TRIF activation induces pain with negligible inflammation, constituting a chronic pain homeostatic mechanism.

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.