PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

Subnormal visual acuity after compliant amblyopia therapy: residual/refractory amblyopia or co-existing pathology? - a retrospective analysis.

Purpose: To assess the prevalence of alternate etiology/co-existing pathology among patients with amblyopia, and to characterize factors contributing to over-diagnosis of amblyopia. Methods: We retrospectively reviewed records of children (from 1 January 2016 to 31 December 2019) who were initially diagnosed as "amblyopia" but later an alternate diagnosis for subnormal vision was established. Patients who had a best corrected visual acuity (BCVA) of ≤20/32 (0.2 logMAR) after compliant amblyopia therapy were divided into 2 groups: those with refractory amblyopia (BCVA improvement from baseline <1 logMAR line) and residual amblyopia (BCVA improvement from baseline >1 logMAR line). Data was collected for presence/absence of amblyogenic risk factors, history, ocular examination, and investigations leading to the final alternate diagnosis. We analyzed the factors that contributed to the initial over-diagnosis of amblyopia using the diagnostic error evaluation and research (DEER) taxonomy tool. Results: During the study period, 508 children with an initial diagnosis of amblyopia met the study criteria. Among these 508 children, 466 were diagnosed to have amblyopia alone, while 26 children (5.1%, median age: 7 years, 17 boys: 9 girls) were revised to have an alternate diagnosis/co-existing pathology. These 26 patients comprised of 2 groups: children referred to us as amblyopia but rediagnosed to have an alternate diagnosis; and a second subset, initially diagnosed by us to have amblyopia, but later found to have alternate diagnosis/co-existing pathology. Subclinical optic neuritis (50%, 13 children), and occult macular dystrophy (OMD) (38.4%, 10 children) were the most frequent alternative diagnoses. Children with ametropic amblyopia (8/26, 30.7%) were most frequently misdiagnosed. Risk factors that led to an initial diagnosis of amblyopia were: high refractive error and heterotropia in 7 patients each (26.9%), anisometropia in 12 (46.1%), and prior pediatric cataract surgery in 4(15.3%). No improvement in BCVA in 21/26 (80.7%) children led to suspicion of co-existing etiology. Other clues were optic disc pallor (11), subnormal color vision (7), history of parental consanguinity in 7, and preceding febrile illness/rhinitis in 1 child. The DEER taxonomy tool suggested that the most common reasons for diagnostic errors were over-emphasis on amblyopia. Conclusion: Our study suggests that 5% of children diagnosed with amblyopia might have co-existing/alternate etiology. Most common co-existing etiologies were subclinical optic neuropathy, and OMD. No improvement in BCVA, subtle history and examination findings prompted further workup. Not considering co-existing etiologies was the most common reason for an initial overdiagnosis of amblyopia.

Cell state transition analysis identifies interventions that improve control of Mycobacterium tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from the tuberculosis (TB) susceptible and resistant mice. We then apply the cSTAR (cell state transition assessment and regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the transcriptional state of tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent Mycobacterium tuberculosis.

PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

The 20/20/20 rule: Practicing pattern and associations with asthenopic symptoms.

Purpose: The present work style and lifestyle have increased the digital device use. Therefore, an increase in digital eyestrain is to be expected. We undertook a survey during coronavirus disease 2019 (COVID-19) pandemic to investigate the practice of 20/20/20 rule and its association with digital device use and asthenopic symptoms. While this rule is commonly advised, little is known about its validity. Methods: An online survey form was disseminated through social media and emails. The questions for eye-related symptoms were similar to the convergence insufficiency symptom survey (CISS). Participants with age ≥5 years were included, with parents completing the survey for children (≤16 years). Results: A total of 432 participants (mean ± standard deviation [SD]: 26.06 ± 13.92 years) were enrolled, of which 125 responses were for children. The 20/20/20 rule was practiced only by 34% of the participants either regularly (n = 38) or occasionally (n = 109). Those who had complaints of burning sensation and headache tended to practice this rule. Among adult participants, more females (47%) practiced this rule when compared to males (23%). Also, adult females significantly (P = 0.04) had more symptoms score when compared to males. In children, no such gender difference was found. Conclusion: Only one-third of participants practice the 20/20/20 rule at least occasionally. More number of adult females being symptomatic and practicing in greater number could be due to higher prevalence of dry eye condition in females. While the symptom of burning sensation could be related to dry eye, that of headache could be related to refractive error or binocular vision dysfunctions.

Cell state transition analysis identifies interventions that improve control of M. tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from TB-susceptible and TB-resistant mice. We then apply the cSTAR (cell State Transition Assessment and Regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the. transcriptional state of the TB-susceptible macrophages towards that of TB-resistant cells. Finally, we demonstrate that the compounds identified with this approach enhance resistance of the TB-susceptible mouse macrophages to virulent M. tuberculosis .

Channeling macrophage polarization by rocaglates increases macrophage resistance to Mycobacterium tuberculosis.

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors.

Sensory perception of environmental cues as a modulator of aging and neurodegeneration: Insights from Caenorhabditis elegans.

Environmental stimuli such as temperature, food, and smell significantly influence the physiology and behavior of animals. Animals are differentially adapted to maintain their internal body functions in response to varied environmental conditions. These external cues are sensed by specialized neurons which are a part of the chemosensory and thermosensory systems. The inability to respond correctly to varied environmental conditions may result in compromised bodily functions and reduced longevity. For example, the ability to sense food is derived from the integrated action of olfactory and gustatory systems. The damage to the olfactory system will affect our decision of palatable food items which in turn can affect the response of the gustatory system, ultimately causing abnormal feeding habits. Recent studies have provided evidence that aging is regulated by sensory perception of environment. Aging is one of the most common causes of various neurodegenerative diseases and the perception of environmental cues is also found to regulate the development of neurodegenerative phenotype in several animal models. However, specific molecular signaling pathways involved in the process are not completely understood. The research conducted on one of the best-studied animal models of aging, Caenorhabditis elegans, has demonstrated multiple examples of gene-environment interaction at the neuronal level which affects life span. The findings may be useful to identify the key neuronal regulators of aging and age-related diseases in humans owing to conserved core metabolic and aging pathways from worms to humans.

The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas.

The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-ß/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.

Visual profile of acquired brain injury in Indian cohort: a retrospective study.

OBJECTIVE: With the increasing global prevalence of acquired brain injury (ABI), the burden of visual problems as a sequelae to ABI is on the rise. This study reports the visual profile of patients with ABI seen in Neuro-Optometry Clinic (NOC) at a tertiary eye-care center in Southern India. METHODS: A retrospective study was carried out between January 2014 and December 2015. Medical records of patients diagnosed with ABI referred by Neuro-Ophthalmologists to the NOC were reviewed. The detailed history, clinical findings of neuro assessment and management details were recorded. RESULTS: Of the 241 patients with ABI, 208 had Traumatic Brain Injury (TBI) and 33 had Cerebro-Vascular Accident (CVA). The mean (SD) age of patients with TBI was 35 ± 14 years and CVA was 52 ± 16 years. Binocular diplopia (61%) was seen predominantly in TBI due to vertical deviation (31%). Cranial nerve palsy was most common in TBI (55%) than CVA (36%) and visual field defects were most frequently seen in CVA (27%). CONCLUSION: Cranial nerve paresis and restrictive strabismus with diplopia were the most common presentations in TBI and visual field defects in CVA. A neuro-optometric evaluation is recommended to identify visual dysfunctions and provide appropriate management options. ABBREVIATIONS: ABI: Acquired Brain Injury; TBI: Traumatic Brain Injury; CVA: Cerebrovascular Accident; NOC: Neuro-Optometry Clinic; NSBVA: Non-Strabismic binocular vision anomalies; OMD: Oculomotor dysfunction; VFD: Visual field defect; GON: Glaucomatous optic neuropathy.

Fine-tuning of macrophage activation using synthetic rocaglate derivatives.

Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

Mycobacteriophage L5Gp56, a novel member of the NrdH family of redoxins.

Mycobacteriophage L5 gene 56 encodes a putative thioredoxin family protein. Phylogenetic analysis revealed that Gp56 and related proteins are distantly related to NrdH - a glutaredoxin homolog which has thioredoxin-like properties. To understand its function, the recombinant version of the protein was biochemically characterized. For the sake of comparison, a mycobacterial thioredoxin, TrxB, was included in the study. Results show that Gp56 can be reduced by dithiothreitol, but only at a higher concentration as compared with TrxB, indicating that the standard redox potential of Gp56 is lower than that of TrxB. The reduced protein can subsequently act as a reductant of protein disulfide bonds. Gp56 can be reduced by NADPH with the help of thioredoxin reductase (TrxR) but less efficiently as compared with TrxB. The abilities of Gp56 and TrxB to reduce Gp50, the L5-encoded ribonucleotide reductase, was examined. While both are capable of executing this function, the former needs more reducing equivalents in the process as compared with the latter. This study shows that L5Gp56 represents a new class of NrdH-like proteins that function optimally in a reducing environment.

The mycobacteriophage D29 gene 65 encodes an early-expressed protein that functions as a structure-specific nuclease.

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is 65, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3' arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3' end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5' and 3' flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophage-derived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.

Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5.

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni, whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages.

Characterization of a galactose binding serum lectin from the Indian catfish, Clarias batrachus: possible involvement of fish lectins in differential recognition of pathogens.

A lectin with molecular mass around 200 kDa was isolated from the serum of the Indian catfish Clarias batrachus. The bioactivity of this serum lectin was Ca2+ and pH dependent. The lectin appeared to be specific for alpha-methyl galactose and sialoglycoproteins like porcine and bovine submaxillary mucin and could agglutinate human, rabbit, mice, rat and chicken erythrocytes. This fish lectin was able to specifically agglutinate different gram negative bacteria. When it was checked against different strains of the fish pathogen Aeromonas sp., it significantly altered the viability and pathogenicity of the bacteria. Binding of the lectin to Aeromonas sp., resulted in a dose dependent increase in the bactericidal activity of fish macrophages. However, when the lectin was checked against different gram positive bacteria it could not agglutinate or affect the viability of those strains and also failed to bring about any significant change in the bactericidal potential of fish macrophages. The lectin was able to induce the proliferation of head kidney lymphocytes of Clarias and helped in the release of 'IL-1' like cytokines from head kidney macrophages.