An optogenetic method for the controlled release of single molecules.

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Roadmap on magnetic nanoparticles in nanomedicine.

Magnetic nanoparticles (MNPs) represent a class of small particles typically with diameters ranging from 1 to 100 nanometers. These nanoparticles are composed of magnetic materials such as iron, cobalt, nickel, or their alloys. The nanoscale size of MNPs gives them unique physicochemical (physical and chemical) properties not found in their bulk counterparts. Their versatile nature and unique magnetic behavior make them valuable in a wide range of scientific, medical, and technological fields. Over the past decade, there has been a significant surge in MNP-based applications spanning biomedical uses, environmental remediation, data storage, energy storage, and catalysis. Given their magnetic nature and small size, MNPs can be manipulated and guided using external magnetic fields. This characteristic is harnessed in biomedical applications, where these nanoparticles can be directed to specific targets in the body for imaging, drug delivery, or hyperthermia treatment. Herein, this roadmap offers an overview of the current status, challenges, and advancements in various facets of MNPs. It covers magnetic properties, synthesis, functionalization, characterization, and biomedical applications such as sample enrichment, bioassays, imaging, hyperthermia, neuromodulation, tissue engineering, and drug/gene delivery. However, as MNPs are increasingly explored for in vivo applications, concerns have emerged regarding their cytotoxicity, cellular uptake, and degradation, prompting attention from both researchers and clinicians. This roadmap aims to provide a comprehensive perspective on the evolving landscape of MNP research.

Characteristics and Prognosis of Infectious Disease Emergencies in Patients with Chronic Kidney Disease in India.

Objectives: Chronic kidney disease (CKD) significantly increases the risk of infectious diseases (IDs), leading to heightened morbidity and mortality. However, there remains a lack of detailed, region-specific studies. This study investigates the clinical spectrum, etiologies, outcomes, and baseline predictors of mortality of ID emergencies in CKD patients in North India. Methods: This retrospective study was conducted at the Postgraduate Institute of Medical Education and Research, Chandigarh, from January 2021 to December 2022. It included patients aged ≥13 years with CKD and IDs admitted to the Acute Care and Emergency Medicine Unit. Results: We enrolled 248 patients (mean age 50 years, 58.1% males). About 60% had CKD stage 5, and 46% were on maintenance hemodialysis. Diabetic kidney disease was the predominant etiology (38.7%). The principal IDs were pneumonia (27.4%), urinary tract infection (UTI) (21.4%), sepsis of unknown primary focus (15.7%), tuberculosis (8.1%), and multisite infections (7.7%). Patients commonly have atypical clinical presentation, e.g., absence of fever and nonspecific symptoms such as shortness of breath and altered mental status. An emergence of multidrug-resistant organisms, e.g., Enterococcus faecium for UTI and Stenotrophomonas maltophilia for catheter-related bloodstream infections, was noted.In-hospital mortality rate was 33.5%, higher with multisite infections (58%) and pneumonia (47%). A low baseline Glasgow coma scale (GCS) was an independent predictor of mortality [odds ratio (OR) 0.786, 95% confidence interval (CI) 0.693-0.891, p-value <0.001]. Conclusion: Effective management and early intervention are needed to improve outcomes in CKD patients with ID emergencies, given the high mortality and atypical clinical presentations. How to cite this article: Prabhahar A, Vijaykumar NA, Selvam S, Ramchandran R, Sethi J, Pannu AK, et al. Characteristics and Prognosis of Infectious Disease Emergencies in Patients with Chronic Kidney Disease in India. Indian J Crit Care Med 2024;28(6):601-606.

Thalamic deep brain stimulation paradigm to reduce consciousness: Cortico-striatal dynamics implicated in mechanisms of consciousness.

Anesthetic manipulations provide much-needed causal evidence for neural correlates of consciousness, but non-specific drug effects complicate their interpretation. Evidence suggests that thalamic deep brain stimulation (DBS) can either increase or decrease consciousness, depending on the stimulation target and parameters. The putative role of the central lateral thalamus (CL) in consciousness makes it an ideal DBS target to manipulate circuit-level mechanisms in cortico-striato-thalamic (CST) systems, thereby influencing consciousness and related processes. We used multi-microelectrode DBS targeted to CL in macaques while recording from frontal, parietal, and striatal regions. DBS induced episodes of abnormally long, vacant staring with low-frequency oscillations here termed vacant, perturbed consciousness (VPC). DBS modulated VPC likelihood in a frequency-specific manner. VPC events corresponded to decreases in measures of neural complexity (entropy) and integration (Φ*), proposed indices of consciousness, and substantial changes to communication in CST circuits. During VPC, power spectral density and coherence at low frequencies increased across CST circuits, especially in thalamo-parietal and cortico-striatal pathways. Decreased consciousness and neural integration corresponded to shifts in cortico-striatal network configurations that dissociated parietal and subcortical structures. Overall, the features of VPC and implicated networks were similar to those of absence epilepsy. As this same multi-microelectrode DBS method-but at different stimulation frequencies-can also increase consciousness in anesthetized macaques, it can be used to flexibly address questions of consciousness with limited confounds, as well as inform clinical investigations of other consciousness disorders.

Predictive Feedback, Early Sensory Representations, and Fast Responses to Predicted Stimuli Depend on NMDA Receptors.

Learned associations between stimuli allow us to model the world and make predictions, crucial for efficient behavior (e.g., hearing a siren, we expect to see an ambulance and quickly make way). While there are theoretical and computational frameworks for prediction, the circuit and receptor-level mechanisms are unclear. Using high-density EEG, Bayesian modeling, and machine learning, we show that inferred "causal" relationships between stimuli and frontal alpha activity account for reaction times (a proxy for predictions) on a trial-by-trial basis in an audiovisual delayed match-to-sample task which elicited predictions. Predictive ß feedback activated sensory representations in advance of predicted stimuli. Low-dose ketamine, an NMDAR blocker, but not the control drug dexmedetomidine, perturbed behavioral indices of predictions, their representation in higher-order cortex, feedback to posterior cortex, and pre-activation of sensory templates in higher-order sensory cortex. This study suggests that predictions depend on alpha activity in higher-order cortex, ß feedback, and NMDARs, and ketamine blocks access to learned predictive information.SIGNIFICANCE STATEMENT We learn the statistical regularities around us, creating associations between sensory stimuli. These associations can be exploited by generating predictions, which enable fast and efficient behavior. When predictions are perturbed, it can negatively influence perception and even contribute to psychiatric disorders, such as schizophrenia. Here we show that the frontal lobe generates predictions and sends them to posterior brain areas, to activate representations of predicted sensory stimuli before their appearance. Oscillations in neural activity (α and ß waves) are vital for these predictive mechanisms. The drug ketamine blocks predictions and the underlying mechanisms. This suggests that the generation of predictions in the frontal lobe, and the feedback pre-activating sensory representations in advance of stimuli, depend on NMDARs.

Topographic organization of connections between prefrontal cortex and mediodorsal thalamus: Evidence for a general principle of indirect thalamic pathways between directly connected cortical areas.

Our ability to act flexibly, according to goals and context, is known as cognitive control. Hierarchical levels of control, reflecting different levels of abstraction, are represented across prefrontal cortex (PFC). Although the mediodorsal thalamic nucleus (MD) is extensively interconnected with PFC, the role of MD in cognitive control is unclear. Tract tracer studies in macaques, involving subsets of PFC areas, have converged on coarse MD-PFC connectivity principles; but proposed finer-grained topographic schemes, which constrain interactions between MD and PFC, disagree in many respects. To investigate a unifying topographic scheme, we performed probabilistic tractography on diffusion MRI data from eight macaque monkeys, and estimated the probable paths connecting MD with each of all 19 architectonic areas of PFC. We found a connectional topography where the orderly progression from ventromedial to anterior to posterolateral PFC was represented from anteromedial to posterolateral MD. The projection zones of posterolateral PFC areas in MD showed substantial overlap, and those of ventral and anteromedial PFC areas in MD overlapped. The exception was cingulate area 24: its projection zone overlapped with projections zones of all other PFC areas. Overall, our data suggest that nearby, functionally related, directly connected PFC areas have partially overlapping projection zones in MD, consistent with a role for MD in coordinating communication across PFC. Indeed, the organizing principle for PFC projection zones in MD appears to reflect the flow of information across the hierarchical, multi-level PFC architecture. In addition, cingulate area 24 may have privileged access to influence thalamocortical interactions involving all other PFC areas.

A Mysterious Case of Pleuroperitoneal Leak Leading to Recurrent Hydrothorax in a Child with Nephrotic Syndrome.

We describe a young girl of steroid resistant nephrotic syndrome with rapidly reaccumulating right sided pleural effusion. On evaluation, the patient was diagnosed with right side pleuro-peritoneal leak leading to recurrent right hydrothorax. To the best of our knowledge, hydrothorax due to transdiaphragmatic shunting in a nephrotic patient has not been reported earlier.

Primate thalamic nuclei select abstract rules and shape prefrontal dynamics.

Flexible behavior depends on abstract rules to generalize beyond specific instances, and outcome monitoring to adjust actions. Cortical circuits are posited to read out rules from high-dimensional representations of task-relevant variables in prefrontal cortex (PFC). We instead hypothesized that converging inputs from PFC, directly or via basal ganglia (BG), enable primate-specific thalamus to select rules. To test this, we simultaneously measured spiking activity across PFC and two connected thalamic nuclei of monkeys applying rules. Abstract rule information first appeared in the ventroanterior thalamus (VA) - the main thalamic hub between BG and PFC. The mediodorsal thalamus (MD) also represented rule information before PFC, which persisted after rule cues were removed, to help maintain activation of relevant posterior PFC cell ensembles. MD, a major recipient of midbrain dopamine input, was first to represent information about behavioral outcomes. This persisted after the trial (also in PFC). A PFC-BG-thalamus model reproduced key findings, and thalamic-lesion modeling disrupted PFC rule representations. These results suggest a revised view of the neural basis of flexible behavior in primates, featuring a central role for thalamus in selecting high-level cognitive information from PFC and implementing post-error behavioral adjustments, and of the functional organization of PFC along its anterior-posterior dimension.

Pseudomonas putida NBRIC19 dihydrolipoamide succinyltransferase (SucB) gene controls degradation of toxic allelochemicals produced by Parthenium hysterophorus.

AIMS: The aim of this study was to identify the gene responsible for degradation of toxic allelochemicals of Parthenium by generating Tn5-induced mutant of Pseudomonas putida NBRIC19. Furthermore, the study characterizes the mutant at physiological, biochemical and molecular level that helped in understanding the mechanisms of reducing the allelopathic inhibition of Parthenium by Ps. putida NBRIC19. METHODS AND RESULTS: Tn5 mutant S-74.3 showing inability to degrade toxic allelochemicals was selected after screening 22 000 transconjugants. Tn5 flanking SucB gene (dihydrolipoamide succinyltransferase) of Ps. putida NBRIC19 was found to be responsible for the degradation of toxic allelochemicals that also affected biofilm formation, chemotaxis and alginate production under toxic environment of allelochemicals. Phenotypic microarray data revealed that the respiratory activity of Ps. putida NBRIC19 and S-74.3 differed on 47 substrates including amino acids, carboxylic acids, peptides and some chemical inhibitors. CONCLUSIONS: Study revealed that SucB gene regulates processes either directly or indirectly in Ps. putida NBRIC19, which on inactivation made the mutant less compatible for tolerating stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first evidence for a functional role of Ps. putida SucB gene in degradation of toxic allelochemicals of Parthenium that lead to reversal of plant growth inhibition by these toxic allelochemicals. The investigation also revealed interesting features about the involvement of microbes in plant-plant allelopathic interactions.

An engineered anisotropic nanofilm with unidirectional wetting properties.

Anisotropic textured surfaces allow water striders to walk on water, butterflies to shed water from their wings and plants to trap insects and pollen. Capturing these natural features in biomimetic surfaces is an active area of research. Here, we report an engineered nanofilm, composed of an array of poly(p-xylylene) nanorods, which demonstrates anisotropic wetting behaviour by means of a pin-release droplet ratchet mechanism. Droplet retention forces in the pin and release directions differ by up to 80 µN, which is over ten times greater than the values reported for other engineered anisotropic surfaces. The nanofilm provides a microscale smooth surface on which to transport microlitre droplets, and is also relatively easy to synthesize by a bottom-up vapour-phase technique. An accompanying comprehensive model successfully describes the film's anisotropic wetting behaviour as a function of measurable film morphology parameters.

Disentangling the influences of multiple thalamic nuclei on prefrontal cortex and cognitive control.

The prefrontal cortex (PFC) has a complex relationship with the thalamus, involving many nuclei which occupy predominantly medial zones along its anterior-to-posterior extent. Thalamocortical neurons in most of these nuclei are modulated by the affective and cognitive signals which funnel through the basal ganglia. We review how PFC-connected thalamic nuclei likely contribute to all aspects of cognitive control: from the processing of information on internal states and goals, facilitating its interactions with mnemonic information and learned values of stimuli and actions, to their influence on high-level cognitive processes, attentional allocation and goal-directed behavior. This includes contributions to transformations such as rule-to-choice (parvocellular mediodorsal nucleus), value-to-choice (magnocellular mediodorsal nucleus), mnemonic-to-choice (anteromedial nucleus) and sensory-to-choice (medial pulvinar). Common mechanisms appear to be thalamic modulation of cortical gain and cortico-cortical functional connectivity. The anatomy also implies a unique role for medial PFC in modulating processing in thalamocortical circuits involving other orbital and lateral PFC regions. We further discuss how cortico-basal ganglia circuits may provide a mechanism through which PFC controls cortico-cortical functional connectivity.

Consciousness depends on integration between parietal cortex, striatum, and thalamus.

The neural substrates of consciousness remain elusive. Competing theories that attempt to explain consciousness disagree on the contribution of frontal versus posterior cortex and omit subcortical influences. This lack of understanding impedes the ability to monitor consciousness, which can lead to adverse clinical consequences. To test substrates and measures of consciousness, we recorded simultaneously from frontal cortex, parietal cortex, and subcortical structures, the striatum and thalamus, in awake, sleeping, and anesthetized macaques. We manipulated consciousness on a finer scale using thalamic stimulation, rousing macaques from continuously administered anesthesia. Our results show that, unlike measures targeting complexity, a measure additionally capturing neural integration (Φ∗) robustly correlated with changes in consciousness. Machine learning approaches show parietal cortex, striatum, and thalamus contributed more than frontal cortex to decoding differences in consciousness. These findings highlight the importance of integration between parietal and subcortical structures and challenge a key role for frontal cortex in consciousness.

Ectopic matrix metalloproteinase-9 expression in human brain tumor cells enhances oncolytic HSV vector infection.

Oncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.

Noncovalent mechanism for the conformal metallization of nanostructured parylene films.

We describe a rapid, reliable method of preparing nanoporous Ni or Co films using nanostructured poly(chloro-p-xylylene) (nanoPPX) films as templates. The nanoPPX films are vapor deposited onto Si substrates using oblique angle polymerization (OAP), resulting in the formation of an obliquely aligned PPX nanorod array on the substrate. The nanoPPX films are then subjected to noncovalent functionalization using an aromatic ligand (i.e., pyridine) by means of treatment with either an aqueous solution of the ligand or ligand vapor. The results of quartz crystal microbalance and X-ray diffraction studies support a model in which pyridine adsorption is facilitated by the formation of pi-pi interactions with aromatic moieties in the amorphous surface regions of nanoPPX. The physisorbed pyridine in the nanoPPX film can subsequently bind a catalytic Pd(II)-based colloidal seed layer. Continuous, conformal Ni or Co films, characterized by FIB/SEM and AFM, are grown on the Pd(II)-laden nanoPPX films using electroless metallization. Analogous metallization of a conventionally deposited planar PPX film results in noncontinuous or patchy metal deposits. Such behavior is attributed to the sluggish adsorption of pyridine in the planar PPX film, resulting in an approximately 22-fold decrease in the quantity of pyridine adsorbed compared to that in a nanoPPX film. Consequently, the level of Pd(II) bound by pyridine on a planar PPX film is insufficient to catalyze continuous metallization. Results of a statistical two-level factorial design indicate that the morphology of the metal layer formed on a nanoPPX film is profoundly influenced by the ligand adsorption condition (i.e., aqueous ligand vs ligand vapor treatment) and is correlated to the catalytic activity of Co films for the production of hydrogen from sodium borohydride decomposition.

Thalamus Modulates Consciousness via Layer-Specific Control of Cortex.

Functional MRI and electrophysiology studies suggest that consciousness depends on large-scale thalamocortical and corticocortical interactions. However, it is unclear how neurons in different cortical layers and circuits contribute. We simultaneously recorded from central lateral thalamus (CL) and across layers of the frontoparietal cortex in awake, sleeping, and anesthetized macaques. We found that neurons in thalamus and deep cortical layers are most sensitive to changes in consciousness level, consistent across different anesthetic agents and sleep. Deep-layer activity is sustained by interactions with CL. Consciousness also depends on deep-layer neurons providing feedback to superficial layers (not to deep layers), suggesting that long-range feedback and intracolumnar signaling are important. To show causality, we stimulated CL in anesthetized macaques and effectively restored arousal and wake-like neural processing. This effect was location and frequency specific. Our findings suggest layer-specific thalamocortical correlates of consciousness and inform how targeted deep brain stimulation can alleviate disorders of consciousness.

CT versus MR Imaging in Estimating Cochlear Radiation Dose during Gamma Knife Surgery for Vestibular Schwannomas.

BACKGROUND AND PURPOSE: Leksell stereotactic radiosurgery is an effective option for patients with vestibular schwannomas. Some centers use a combination of stereotactic CT fused with stereotactic MR imaging to achieve an optimal target definition as well as minimize the radiation dose delivered to adjacent structures that correlate with hearing outcomes. The present prospective study was designed to determine whether there is cochlear dose variability between MR imaging and CT. MATERIALS AND METHODS: Fifty consecutive patients underwent stereotactic radiosurgery for vestibular schwannomas. Dose-planning was performed using high-definition fused stereotactic MR imaging and stereotactic CT images. The 3D cochlear volume was determined by delineating the cochlea on both CT and T2-weighted MR imaging. The mean radiation dose, maximum dose, and 3- and 4.20-Gy cochlear volumes were identified using standard Leksell Gamma Knife software. RESULTS: The median mean radiation dose delivered to the cochlea was 3.50 Gy (range, 1.20-6.80 Gy) on CT and 3.40 Gy (range, 1-6.70 Gy) on MR imaging (concordance correlation coefficient = 0.86, r 2 = 0.9, P ≤ .001). The median maximum dose delivered to the cochlea was 6.7 Gy on CT and 6.6 Gy on MR imaging (concordance correlation coefficient = 0.89, r 2 = 0.90, P ≤ .001). Dose-volume histograms generated from CT and MR imaging demonstrated a strong level of correlation in estimating the 3- and 4.20-Gy volumes (concordance correlation coefficient = 0.81, r 2 = 0.82, P ≤ .001 and concordance correlation coefficient = 0.87, r 2 = 0.89, P ≤ .001). CONCLUSIONS: Both MR imaging and CT provide similar cochlear dose parameters. Despite the reported superiority of CT in identifying bony structures, high-definition MR imaging alone is sufficient to identify the radiation doses delivered to the cochlea.

A Subcortical Pathway for Rapid, Goal-Driven, Attentional Filtering.

Prefrontal cortex can exercise goal-driven attentional control over sensory information via cortical pathways. However, recent work demonstrates that prefrontal cortex can also influence thalamic relay nuclei via the thalamic reticular nucleus. This suggests the prefrontal-thalamic pathway mediates rapid and goal-driven attentional filtering at the earliest stages of sensory processing.

Troxerutin, a plant flavonoid, protects cells against oxidative stress-induced cell death through radical scavenging mechanism.

Troxerutin (TRX) is a flavonoid present in tea, coffee, cereal grains, various fruits and vegetables have been reported to exhibit radioprotective, antithrombotic, nephro and hepato-protective effects. A systematic study was undertaken to evaluate its free radical scavenging ability and anti-apoptotic activity in cell-free and cellular systems. TRX scavenged superoxide, nitric oxide and also other model stable radicals like 1,1-diphenyl-2-picryl-hydazyl, and 2,2'-azinobis3-ethylbenzothiazoline-6-sulfonic acid. It reacted with hydroxyl radicals, carbonate and thiocyanate anions, as evaluated by pulse radiolysis and stopped flow techniques. TRX protected different cell types (epithelial cells, fibroblasts and lymphocytes) against peroxyl radical-induced apoptosis, necrosis and mitotic death. It scavenged intracellular basal and inducible ROS levels and also restored depletion of intracellular GSH levels, suggesting that free radical scavenging ability may be responsible for the observed cytoprotection of different cell types. TRX may find application as an adjuvant in treating various diseases attributed to oxidative stress.

Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.