Imaging of DNA and RNA in Living Eukaryotic Cells to Reveal Spatiotemporal Dynamics of Gene Expression.

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.

Intracellular mRNA transport and localized translation.

Fine-tuning cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression. High-resolution imaging technologies to detect mRNAs and their translation state have revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, enabling cells to tune gene expression locally. Therefore, mRNA localization is a conserved and integral part of gene expression regulation from prokaryotic to eukaryotic cells. In this Review, we discuss the mechanisms of mRNA transport and local mRNA translation across the kingdoms of life and at organellar, subcellular and multicellular resolution. We also discuss the properties of messenger ribonucleoprotein and higher order RNA granules and how they may influence mRNA transport and local protein synthesis. Finally, we summarize the technological developments that allow us to study mRNA localization and local translation through the simultaneous detection of mRNAs and proteins in single cells, mRNA and nascent protein single-molecule imaging, and bulk RNA and protein detection methods.

Real-time imaging of Arc/Arg3.1 transcription ex vivo reveals input-specific immediate early gene dynamics.

The ability of neurons to process and store salient environmental features underlies information processing in the brain. Long-term information storage requires synaptic plasticity and regulation of gene expression. While distinct patterns of activity have been linked to synaptic plasticity, their impact on immediate early gene (IEG) expression remains poorly understood. The activity regulated cytoskeleton associated (Arc) gene has received wide attention as an IEG critical for long-term synaptic plasticity and memory. Yet, to date, the transcriptional dynamics of Arc in response to compartment and input-specific activity is unclear. By developing a knock-in mouse to fluorescently tag Arc alleles, we studied real-time transcription dynamics after stimulation of dentate granule cells (GCs) in acute hippocampal slices. To our surprise, we found that Arc transcription displayed distinct temporal kinetics depending on the activation of excitatory inputs that convey functionally distinct information, i.e., medial and lateral perforant paths (MPP and LPP, respectively). Moreover, the transcriptional dynamics of Arc after synaptic stimulation was similar to direct activation of GCs, although the contribution of ionotropic glutamate receptors, L-type voltage-gated calcium channel, and the endoplasmic reticulum (ER) differed. Specifically, we observed an ER-mediated synapse-to-nucleus signal that supported elevations in nuclear calcium and, thereby, rapid induction of Arc transcription following MPP stimulation. By delving into the complex excitation-transcription coupling for Arc, our findings highlight how different synaptic inputs may encode information by modulating transcription dynamics of an IEG linked to learning and memory.

Glutamate-induced RNA localization and translation in neurons.

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single ß-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of ß-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling.

Polarized Rac1 signaling is a hallmark of many cellular functions, including cell adhesion, motility, and cell division. The two steps of Rac1 activation are its translocation to the plasma membrane and the exchange of nucleotide from GDP to GTP. It is, however, unclear whether these two processes are regulated independent of each other and what their respective roles are in polarization of Rac1 signaling. We designed a single-particle tracking (SPT) method to quantitatively analyze the kinetics of Rac1 membrane translocation in living cells. We found that the rate of Rac1 translocation was significantly elevated in protrusions during cell spreading on collagen. Furthermore, combining FRET sensor imaging with SPT measurements in the same cell, the recruitment of Rac1 was found to be polarized to an extent similar to that of the nucleotide exchange process. Statistical analysis of single-molecule trajectories and optogenetic manipulation of membrane lipids revealed that Rac1 membrane translocation precedes nucleotide exchange, and is governed primarily by interactions with phospholipids, particularly PI(3,4,5)P3, instead of protein factors. Overall, the study highlights the significance of membrane translocation in spatial Rac1 signaling, which is in addition to the traditional view focusing primarily on GEF distribution and exchange reaction.

Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Access to chronic kidney disease (CKD) care: Its barriers and facilitators in a community development block in Purba Bardhaman, West Bengal: A qualitative study.

Background: Chronic kidney disease (CKD), a leading public health problem, has potential risks and serious implications on the health of individuals and society at large. Few studies explored the factors that influence access to CKD care at the community level in rural areas of West Bengal. A qualitative study was planned among key stakeholders to explore their views and experiences and to identify the barriers and potential facilitators that influence access to CKD care at the primary care level. Methods: Total 23 stakeholders participated in the study. Seventeen in-depth interviews (IDIs) were conducted on a purposive sample of stakeholders (CKD patients, healthcare providers (HCPs) and health planners) and one focus group discussion (FGD) among six community health workers. The audio-recorded interviews were transcribed verbatim. The Lévesque's framework for access to care as modified by Jodie Bailie et al. was employed to construct interview guides and structure the initial codes. Thematic analysis was undertaken using QSR NVivo version 11 using both inductive and deductive approaches. Results: The major barriers to CKD care at patient level were poor knowledge and awareness of CKD and at the health system level was shortage of skilled staffs, diagnostics and medicines and fragmented referrals. The potential facilitators identified were educational activities to increase the awareness of CKD among HCPs and patients; provision of CKD-related supplies and a system-level approach to care coordination along with m-health-based care. Conclusions: Targeted CKD screening programs and CKD specific trainings may improve awareness of CKD. Additionally, stronger primary care infrastructure, availability of essential drugs and diagnostics and creating an efficient referral process for the quality CKD care are the need of the hour.

An Unexpected Cause of Syncope.

Syncope is a common chief complaint among patients presenting to the emergency department, the etiology of which can often be discerned with a thorough history and physical examination. Inversely, liposarcomas are rare tumors that frequently pose a diagnostic challenge as the clinical presentation is highly nonspecific and varies greatly depending on the anatomic location and size of the tumor. Here we present a case of retroperitoneal liposarcomas (RLS) presenting to the emergency department (ED) with a sole complaint of syncope, resulting in a diagnostic dilemma. This clinical scenario highlights the significance of thorough physical examination regardless of the presenting chief complaint, as unexpected physical examination findings prompted an extended work-up and thus facilitated the diagnosis, providing the opportunity for early intervention and resection of the tumor.

Maintenance of a short-lived protein required for long-term memory involves cycles of transcription and local translation.

Activity-dependent expression of immediate early genes (IEGs) is critical for long-term synaptic remodeling and memory. It remains unknown how IEGs are maintained for memory despite rapid transcript and protein turnover. To address this conundrum, we monitored Arc, an IEG essential for memory consolidation. Using a knockin mouse where endogenous Arc alleles were fluorescently tagged, we performed real-time imaging of Arc mRNA dynamics in individual neurons in cultures and brain tissue. Unexpectedly, a single burst stimulation was sufficient to induce cycles of transcriptional reactivation in the same neuron. Subsequent transcription cycles required translation, whereby new Arc proteins engaged in autoregulatory positive feedback to reinduce transcription. The ensuing Arc mRNAs preferentially localized at sites marked by previous Arc protein, assembling a "hotspot" of translation, and consolidating "hubs" of dendritic Arc. These cycles of transcription-translation coupling sustain protein expression and provide a mechanism by which a short-lived event may support long-term memory.

Prevalence and correlates of depression, anxiety, and stress among high school students in a block of Hooghly district, West Bengal: Across-sectional study.

BACKGROUND: Adolescent mental health is a concern. A high magnitude of deranged mental health conditions among adolescents is prevailing, which often gets extended into adulthood too. Hence, assessment of mental health morbidities like depression, anxiety, and stress is crucial to address them. This study aimed to estimate the prevalence of depression, anxiety, and stress among school-going adolescents studying in classes IX-XII and find out its correlates among the subjects. MATERIALS AND METHODS: This cross-sectional study was conducted among 812 adolescents studying in classes IX-XII in four randomly selected schools of Haripal block of Hooghly district, West Bengal, in 2022. A complete enumeration of students in each class of the selected schools was done. Data were collected by using a pretested schedule that included standardized depression, anxiety, and stress scale (DASS-21). Descriptive statistics and a Chi-square test were applied. Regression analysis was doneto determine associations and compute the adjusted odds ratio. Data were analyzed using SPSS version 23. RESULTS: Overall prevalence of depression, anxiety, and stress were found to be 52.3%, 47.4%, and 33.7% respectively. Subscale scores shows 16.1% and 10.8% suffered from extremely severe depression and anxiety, respectively. It was noted that these were more common among female students, the late adolescent age group (16-19 years), students having social problems in the family, and those reporting using substances. CONCLUSION: The prevalence of depression, anxiety, and stress being high among school students indicates a significant burden of mental health disease. Effective strategies to alleviate the adverse mental health, along with early identification of disease, can help in along way.

Sphingomonas paucimobilis bacteremia and tricuspid valve endocarditis in a patient with intravenous drug use.

Sphingomonas paucimobilis is an aerobic, yellow-pigmented, glucose non-fermenting, gram negative bacillus that is a rare cause of human infection found mostly in the immunocompromised and also in intravenous (IV) drug users. We report a case of a 31-year-old female with current IV drug use, who presented with chest pain and was diagnosed with tricuspid valve endocarditis with S. paucimobilis bacteremia and pulmonary infarction of the right middle lobe. The patient initially presented with sharp right sided chest pain. She was treated with meropenem and levofloxacin based on the susceptibility profile. Our purpose is to highlight the treatment options and raise awareness of this uncommon organism. Even though Sphingomonas is considered to be of low-pathogenicity, it can be fatal if not treated properly and not diagnosed early.

Severe Thrombocytopenia Associated with Dengue Fever: An Evidence-Based Approach to Management of Thrombocytopenia.

Dengue is a mosquito-borne viral illness common in tropical and subtropical countries but very rare in the United States. Patients infected with dengue often present with thrombocytopenia. In the setting of dengue, platelet transfusions as a treatment for thrombocytopenia have no clear benefits in reduction of severe bleeding or improvement of the platelet count. Here, we present a case of a traveler infected with dengue virus and discuss the approach to treat thrombocytopenia.

A Unique Presentation of Acute Kidney Injury With COVID-19.

Although the respiratory system is the primary target of COVID-19 pneumonia, it can also notably affect the other systemic organs such as renal and cardiac. The incidence and prevalence of SARS CoV-2 associated acute renal failure are emerging day by day. While the pathogenesis is not clearly understood, it is considered multifactorial. Initially, the COVID-19-associated renal dysfunction was limited to acute tubular injury. However, over time a wide spectrum of clinical manifestations has been reported. Therefore, prompt investigation and early initiation of supportive treatment can potentially reduce the mortality and morbidity associated with this systemic disease. In this case report, we present a unique presentation of a COVID-19 with acute kidney injury where the patient was admitted to the intensive care unit with clinical features of acute renal failure with concomitant diagnosis of COVID-19, unlike other reported cases where patients were admitted to the intensive unit with respiratory distress and subsequently developed renal failure.

Non-aromatic Anticonvulsant (Divalproex Sodium)-Induced Drug Reaction With Eosinophilia and Systemic Symptoms (DRESS) Syndrome.

A wide array of commonly prescribed antiepileptic medications, antibiotics, antivirals, anti-parasitic, and antihyperuricemic can cause Drug Reaction Eosinophilia and Systemic Syndrome (DRESS)- a drug induced hypersensitivity reaction characterized by cutaneous manifestation, fever, eosinophilia, thrombocytopenia and one or more visceral organ involvement. The rare occurrence in clinical settings and wide variety of clinical presentations make DRESS a diagnostically challenging case. A vast majority of DRESS cases are attributed to the most commonly prescribed anticonvulsant medications - phenytoin and carbamazepines. Even though non-aromatic divalproex sodium/valproic acid (VPA) can cause life-threatening fatal hypersensitivity reactions on rare occasions, a handful cases of valproate-induced DRESS have been reported. We hereby report a case of a 57-year-old cognitively impaired person with polypharmacy who presented with worsening diffuse skin rash, fever, dysphagia, eosinophilia, thrombocytopenia, and mixed type of hepatic injury. The patient was eventually diagnosed with DRESS due to divalproex sodium- an anticonvulsant medication. The objective of our report is to identify and recognize the rare yet proper causative agent that induces DRESS and potential mitigation of significant systemic consequences by its subsequent withdrawal.

Spatiotemporal Insights Into RNA-Organelle Interactions in Neurons.

Neurons exhibit spatial compartmentalization of gene expression where localization of messenger RNAs (mRNAs) to distal processes allows for site-specific distribution of proteins through local translation. Recently, there have been reports of coordination between mRNA transport with vesicular and organellar trafficking. In this review, we will highlight the latest literature on axonal and dendritic local protein synthesis with links to mRNA-organelle cotransport followed by emerging technologies necessary to study these phenomena. Recent high-resolution imaging studies have led to insights into the dynamics of RNA-organelle interactions, and we can now peer into these intricate interactions within subcellular compartments of neurons.

Critical role of lipid rafts in virus entry and activation of phosphoinositide 3' kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells.

Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3'-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl ß-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3'-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection.

Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation.

BACKGROUND: Activation of microglia, the resident macrophages of the central nervous system (CNS), is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB) is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4), one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. METHODS: For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS). Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs). Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA) were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. RESULTS: LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown. We found that Klf4 can potentially interact with pNF-κB and is important for iNOS and Cox-2 promoter activity in vitro. CONCLUSIONS: These studies demonstrate the role of Klf4 in microglia in mediating neuroinflammation in response to the bacterial endotoxin LPS.

Uncommon Coronary Abnormalities in a Patient with Discoid Lupus Erythematosus.

A 43-year-old male presented with shortness of breath and palpitations. Physical examination was significant for skin lichenification, an erythematous maculopapular rash with annular plaques, a fissured tongue, and digital clubbing. Electrocardiogram captured a supraventricular tachycardia and right bundle branch block. Left heart catheterization found several proximal left coronary artery branch aneurysms, a right coronary-pulmonary artery fistula, venous-luminal vessels between the right heart chambers and cardiac veins, and thebesian venous lakes. Functional cardiac capacity was reduced, with left ventricular ejection fraction of 35 to 40%, on ventriculogram. A diagnosis of discoid lupus erythematosus was established after skin biopsies revealed systemic lupus erythematosus-like features but negative anti-nuclear, anti-Smith, anti-ds-deoxyribonucleic acid, anti-Ro, and anti-La antibodies. The case reported is unique, in that while our patient had pathologic electrophysiologic changes typically seen with autoimmune disease, the patient had many anatomical cardiac anomalies without atherosclerotic coronary disease. It is uncertain whether these findings are purely incidental or are related to an underlying genetic disorder.

Tobacco carcinogen induces microglial activation and subsequent neuronal damage.

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific procarcinogen. We have investigated whether NNK causes inflammatory upheaval in the brain by activation of resident microglia and astrocyte and result in bystander neuronal damage. We have carried out the work in both in vitro and in vivo models. We have found that treatment with NNK causes significant activation of mouse microglial (BV2) cell line as evident by increase in reactive oxygen species and nitric oxide level. Western blot analysis has showed increase in proinflammatory signaling proteins, proinflammatory effector proteins, and other stress-related proteins. Interestingly, increased levels of proinflammatory cytokines like interleukin (IL)-6, tumor necrosis factor-alpha, monocyte chemoattractant protein 1 (MCP1), and IL-12p70 are also detected. Work from our in vivo studies has demonstrated similar increase in proinflammatory signaling and effector molecules along with the proinflammatory cytokine levels, following NNK treatment. Immunohistochemical staining of the brain sections of NNK-treated mice reveals massive microglial and astrocyte activation along with distinct foci of neuronal damage. Both in vitro and in vivo results provide strong indication that NNK causes significant upheaval of the inflammatory condition of brain and inflicts subsequent neuronal damage.