Melding Synthetic Molecules and Genetically Encoded Proteins to Forge New Tools for Neuroscience.

Unraveling the complexity of the brain requires sophisticated methods to probe and perturb neurobiological processes with high spatiotemporal control. The field of chemical biology has produced general strategies to combine the molecular specificity of small-molecule tools with the cellular specificity of genetically encoded reagents. Here, we survey the application, refinement, and extension of these hybrid small-molecule:protein methods to problems in neuroscience, which yields powerful reagents to precisely measure and manipulate neural systems.

Lipid Expansion Microscopy.

Strategies to visualize cellular membranes with light microscopy are restricted by the diffraction limit of light, which far exceeds the dimensions of lipid bilayers. Here, we describe a method for super-resolution imaging of metabolically labeled phospholipids within cellular membranes. Guided by the principles of expansion microscopy, we develop an all-small molecule approach that enables direct chemical anchoring of bioorthogonally labeled phospholipids into a hydrogel network and is capable of super-resolution imaging of cellular membranes. We apply this method, termed lipid expansion microscopy (LExM), to visualize organelle membranes with precision, including a unique class of membrane-bound structures known as nuclear invaginations. Compatible with standard confocal microscopes, LExM will be widely applicable for super-resolution imaging of phospholipids and cellular membranes in numerous physiological contexts.

Modular Enzyme- and Light-Based Activation of Cyclopropene-Tetrazine Ligation.

We describe a modular activation strategy for cyclopropene-tetrazine ligation. This activation strategy uses chemically diverse enzyme- or photolabile protecting groups as cyclopropene reactivity cages. The linkages between the caging groups and cyclopropene are through carbamates, thus permitting the application of diverse cages to allow bioorthogonal reactivity by administering enzymes or light.

Bioproduction of fumaric acid: an insight into microbial strain improvement strategies.

Fumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydrate derived platform chemical. Currently, it is commercially sourced from petrochemicals by chemical conversion. The shift to biochemical synthesis has become essential for sustainable development and for the transition to a biobased economy from a petroleum-based economy. The main limitation is that the concentrations of FA achieved during bioproduction are lower than that from a chemical process. Moreover, the high cost associated with bioproduction necessitates a higher yield to improve the feasibility of the process. To this effect, genetic modification of microorganism can be considered as an important tool to improve FA yield. This review discusses various genetic modifications strategies that have been studied in order to improve FA production. These strategies include the development of recombinant strains of Rhizopus oryzae, Escherichia coli, Saccharomyces cerevisiae, and Torulopsis glabrata as well as their mutants. The transformed strains were able to accumulate fumaric acid at a higher concentration than the corresponding wild strains but the fumaric acid titers obtained were lower than that reported with native fumaric acid producing R. oryzae strains. Moreover, one plausible adoption of gene editing tools, such as Agrobacterium-mediated transformation (AMT), CRISPR CAS-9 and RNA interference (RNAi) mediated knockout and silencing, have been proposed in order to improve fumaric acid yield. Additionally, the introduction of the glyoxylate pathway in R. oryzae to improve fumaric acid yield as well as the biosynthesis of fumarate esters have been proposed to improve the economic feasibility of the bioprocess. The adoption of some of these genetic engineering strategies may be essential to enable the development of a feasible bioproduction process.

Potential of biological approaches for cyanotoxin removal from drinking water: A review.

Biological treatment of cyanotoxins has gained much importance in recent decades and holds a promise to work in coordination with various physicochemical treatments. In drinking water treatment plants (DWTPs), effective removal of cyanotoxins with reduced toxicity is a primary concern. Commonly used treatments, such as ozonation, chlorination or activated carbon, undergo significant changes in their operating conditions (mainly dosage) to counter the variation in different environmental parameters, such as pH, temperature, and high cyanotoxin concentration. Presence of metal ions, natural organic matter (NOM), and other chemicals demand higher dosage and hence affect the activation energy to efficiently break down the cyanotoxin molecule. Due to these higher dose requirements, the treatment leads to the formation of toxic metabolites at a concentration high enough to break the guideline values. Biological methods of cyanotoxin removal proceed via enzymatic pathway where the protein-encoding genes are often responsible for the compound breakdown into non-toxic metabolites. However, in contrast to the chemical treatment, the biological processes advance at a much slower kinetic rate, predominantly due to a longer onset period (high lag phase). In fact, more than 90% of the studies reported on the biological degradation of the cyanotoxins attribute the biodegradation to the bacterial suspension. This suspended growth limits the mass transfer kinetics due to the presence of metal ions, NOMs and, other oxidizable matter, which further prolongs the lag phase and makes biological process toxic-free, albeit less efficient. In this context, this review attempts to bring out the importance of the attached growth mechanism, in particular, the biofilm-based treatment approaches which can enhance the biodegradation rate.

Single-Acquisition Triple-Bolus Dual-Energy CT Protocol for Comprehensive Evaluation of Renal Masses: A Single-Center Randomized Noninferiority Trial.

OBJECTIVE: The primary objective of this study was to compare triple-bolus dual-energy CT (DECT) against standard triple-phase MDCT in terms of appropriateness of patient treatment. SUBJECTS AND METHODS: One hundred twenty-four patients with suspected renal masses seen at ultrasound were randomized into triple-bolus DECT and triple-phase MDCT groups. Patients in the triple-bolus DECT group underwent synchronous corticomedullary nephrographic delayed-phase triple-bolus DECT. In the triple-phase MDCT group, single-energy triple-phase scans were acquired after an unenhanced scan. The primary outcome was appropriateness of treatment received at 1 year. The predefined noninferiority limit was 10%. Histopathologic analysis or follow-up confirmed the benign or malignant nature of the masses. Diagnostic accuracy to differentiate benign from malignant masses was calculated. Size-specific dose estimates were compared. RESULTS: After excluding six patients, 118 patients were analyzed (62 triple-bolus DECT; 56 triple-phase MDCT). Treatment appropriateness was not significantly different (p = 0.9397) between the two groups (61/62 [98.39%; 95% CI, 95.26-101.52%] for triple-bolus DECT vs 55/56 [98.21%; 95% CI, 94.74-101.68%] for triple-phase MDCT). The absolute difference was 0.18% (95% CI, -4.48% to 4.84%). Both techniques had similar diagnostic accuracy (sensitivity, 98.25% vs 96.67%; specificity, 98.17% vs 97.97%). The mean (± SD) size-specific dose estimate was significantly lower for triple-bolus DECT than for triple-phase MDCT (19.02 ± 4.07 vs 57.04 ± 15.17 mGy; p < 0.0001). CONCLUSION: Single-acquisition triple-bolus DECT is noninferior to triple-phase MDCT, with similar diagnostic accuracy but delivering significantly less radiation.

Inexpensive multigram-scale synthesis of cyclic enamines and 3-N spirocyclopropyl systems.

Cyclic enamines are important synthons for many synthetic and pharmacological targets. Here, we report an inexpensive, catalyst-free, multigram-scale synthesis for cyclic enamines with exocyclic double bonds and four- to seven-membered rings. This strategy is more conducive to scale up, permissive of functionalization around the cyclic system, and less sensitive to the nature of the N-protecting group than previously-described methods for cyclic enamine synthesis. Further, we explore application of these enamines to the synthesis of highly-strained spirocyclic 3N-cyclopropyl scaffolds.

Caged cyclopropenes for controlling bioorthogonal reactivity.

Bioorthogonal ligations have been designed and optimized to provide new experimental avenues for understanding biological systems. Generally, these optimizations have focused on improving reaction rates and orthogonality to both biology and other members of the bioorthogonal reaction repertoire. Less well explored are reactions that permit control of bioorthogonal reactivity in space and time. Here we describe a strategy that enables modular control of the cyclopropene-tetrazine ligation. We developed 3-N-substituted spirocyclopropenes that are designed to be unreactive towards 1,2,4,5-tetrazines when bulky N-protecting groups sterically prohibit the tetrazine's approach, and reactive once the groups are removed. We describe the synthesis of 3-N spirocyclopropenes with an appended electron withdrawing group to promote stability. Modification of the cyclopropene 3-N with a bulky, light-cleavable caging group was effective at stifling its reaction with tetrazine, and the caged cyclopropene was resistant to reaction with biological nucleophiles. As expected, upon removal of the light-labile group, the 3-N cyclopropene reacted with tetrazine to form the expected ligation product both in solution and on a tetrazine-modified protein. This reactivity caging strategy leverages the popular carbamate protecting group linkage, enabling the use of diverse caging groups to tailor the reaction's activation modality for specific applications.

Lipidated cyclopropenes via a stable 3-N spirocyclopropene scaffold.

Lipidated cyclopropenes serve as useful bioorthogonal reagents for imaging cell membranes due to the cyclopropene's small size and ability to ligate with pro-fluorescent tetrazines. Previously, the lipidation of cyclopropenes required modification at the C3 position because methods to append lipids at C1/C2 were not available. Herein, we describe C1/C2 lipidation with the biologically active lipid ceramide and a common phospholipid using a cyclopropene scaffold whose reactivity with 1,2,4,5-tetrazines has been caged.

Synthesis of OSL nanophosphor Li3B7O12:Mn and its dosimetric properties.

The present paper reports the structural, morphological and optical properties of nanophosphor Li3B7O12:Mn with an optimised dopant concentration of 0.25 mol% and its surface modification under the irradiation of 250 keV proton beams and gamma photons for ion fluence ranging from 1 × 1013 to 6.25 × 1015 ions cm-2 and doses from 100 mGy-100 Gy, respectively. This nanophosphor has been synthesised by the high temperature solid state reaction method. Its optical properties are characterised by optically stimulated luminescence (OSL) and thermo luminescence (TL) techniques. This nanophosphor is polycrystalline in nature with a grain size of 40-80 nm confirmed by x-ray diffraction (XRD) and transmission electron microscopy (TEM). The OSL decay and TL glow curve response of the proton beam irradiated samples exhibit significant intensity at a fluence of 2.5 × 1014 ions cm-2. Moreover, Li3B7O12:Mn displays a linear response for gamma doses in the range of 100 mGy-50 Gy. We have also investigated the reusability and reproducibility of this material. The above study demonstrates that Li3B7O12:Mn is a robust and promising candidate for medical proton dosimetry.

A photocaged, cyclopropene-containing analog of the amino acid neurotransmitter glutamate.

Substituted cyclopropenes serve as compact biorthogonal appendages that enable analysis of biomolecules in complex systems. Neurotransmitters, a chemically diverse group of biomolecules that control neuron excitation and inhibition, are not among the systems that have been studied using biorthogonal chemistry. Here we describe the synthesis of cyclopropene-containing analogs of the excitatory amino acid neurotransmitter glutamate starting from a Garner's aldehyde-derived alkyne. The deprotected cyclopropene glutamate was stable in solution but decomposed upon concentration. Appending a light-cleavable group improved the stability of the cyclopropene while simultaneously caging the neurotransmitter. This strategy has the potential to permit deployment of cyclopropene-modified glutamate as a bioorthogonal probe of the neurotransmitter glutamate in vivo with spatiotemporal precision.

Nasopharyngeal Foreign Body with Unusual Presentation.

Introduction: An unusual nasopharyngeal foreign body in a very young child with no clinical symptoms is a rare case presentation. Case Report: A nine-month-old child presented with a suspected history of foreign body ingestion without any clue to the parents about the nature of the foreign body. X-ray of the nasopharynx revealed a sharp unusual metallic "Louis Vuitton" shoe logo that the child had accidentally inserted into the nasopharynx via the oral cavity while playing. Foreign body was removed under general anesthesia without complications. Conclusion: X-ray nasopharynx should be included in the examination of a suspected case of foreign body ingestion, as an unusual shape of foreign body can even produce no clinical symptoms but still pose a potential life threat due to its dislodgement into the airway if missed or delayed.

Risk Factors Associated with Mortality in Patients with Mucormycosis Post Severe Acute Respiratory Syndrome Coronavirus-2 (Sars-Cov-2) Infection.

To study the potential risk factors associated with mortality in patients with mucormycosis. A retrospective study of 490 patients with diagnosis of sinonasal mucormycosis was done. They were divided in two groups-Group A included 87 patients that expired during the study period and Group B included 403 control patients. All the demographic, clinical and outcome parameters were collected from the patient's record files and noted in a structured case proforma and were analysed. During the mean hospital stay of 22 ± 6 days, 17.7% patients expired during the treatment course. Multiple risk factors like uncontrolled diabetes, dyselectrolytemia, underlying renal disease and extensive nature of the disease involving orbit, intracranium and with pulmonary dissemination, all were associated with high mortality in Mucormycosis. Early recognition and appropriate management of the secondary factors can grossly reduce the risk of mortality in patients with mucormycosis.

Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore.

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

Secondary Cutaneous Mucormycosis - Retrospective Analysis From Tertiary Care Hospitall.

Background: Covid-19 infection increases the risk of opportunistic infections like mucormycosis. Cutaneous mucormycosis can occur primarily by direct inoculation or secondary to involvement of the underlying structures. Cutaneous manifestations include tender, erythematous, indurated lesions and necrotic plaques. As the disease evolves, cutaneous features manifest progressively. Objectives: To study the manifestations of the cutaneous signs of sinonasal mucormycosis and management of such cases based on severity of involvement. Materials and methods: A retrospective analysis of 21 patients with diagnosis of cutaneous mucormycosis secondary to sinonasal involvement was done with assessment of their skin lesion, area involved and their clinical stage being noted at the time of admission and after 24 h. Treatment consisted of combination of surgical debridement, daily dressing and liposomal amphotericin B. Observations and Results: Out of total 21 patients, there were 10 males and 11 females. Among risk factors, 14 cases had history of covid 19 infection, 5 had history of steroid intake, 6 had history of ICU stay and all had deranged blood sugar levels. Among disease prognosis, excellent outcomes appeared in stage I and stage III showed worst outcome. Conclusion: Since initial clinical presentation is similar to cellulitis and other soft-tissue infections, early recognition is difficult. In this cohort, the prognosis of secondary cutaneous mucormycosis remained poor, especially in ICU patients and those with numerous predisposing factors. Such patients presented in late stages of the disease and mortality rate was very high in such group.

Normocalcemic Parathyroid Adenoma with Brown's Tumor Maxilla: A Rare Case.

Introduction: Primary hyperparathyroidism due to parathyroid adenoma commonly causes raised serum calcium and focal giant cell lytic lesions in bones known as Brown's tumors. It is more common in females in the post-menopausal age group. Case Report: We report a case of a 29-year-old female patient with Brown's tumor maxilla in a clinical setting of normocalcemic primary hyperparathyroidism. The patient presented to us with facial and palatal swelling for which FNAC was done. Cytology revealed hemosiderin-laden macrophages suspicious for Brown's tumor. On further imaging studies such as CT Neck, Tc99 Sestamibi scan, and other biochemical tests like parathyroid hormone assay and serum calcium level, the diagnosis of a hyperfunctioning parathyroid gland with normal calcium level was made. Parathyroidectomy was performed and parathyroid adenoma came out to be the primary pathology. On post-operative follow up there was regression of the swelling on the face and palate relieving the patient symptomatically. Conclusion: The diagnostic suspicion of primary hyperparathyroidism should be kept in mind whenever a young female presents with suspected Brown's tumor, even with normal serum calcium levels, for appropriate management. Ours was a highly uncommon case that was a diagnostic challenge and had a successful treatment outcome. Very few such cases have been reported in the literature to date to the best of our knowledge.

Diabetic Retinopathy: Clinical Features, Risk Factors, and Treatment Options.

Diabetic retinopathy is a common complication of diabetes that affects the eyes and can lead to severe vision loss or blindness if left untreated. Chronic hyperglycemia destroys the blood vessels in the retina, resulting in diabetic retinopathy. The damage can lead to leakage of fluid and blood into the retina, causing edema, hemorrhages, and ischemia. A thorough evaluation by an ophthalmologist is necessary to determine the most appropriate course of treatment for each patient with diabetic retinopathy. The article discusses various surgical treatment options for diabetic retinopathy, including vitrectomy, scleral buckling, epiretinal membrane peeling, retinal detachment repair, and the risk factors of diabetic retinopathy. These surgical techniques can help to address the underlying causes of vision loss and prevent further complications from developing or worsening. To avoid complications and maintain vision, this review emphasizes the significance of early detection and treatment of diabetic retinopathy. Patients with diabetic retinopathy can improve their eyesight and quality of life with the help of some surgical treatments. The article also highlights some case studies in the field of diabetic retinopathy.

Comparative Study of Inlay and Overlay Cartilage Perichondrium Composite Graft Myringoplasty.

To compare results of inlay and overlay cartilage-perichondrium composite graft myringoplasty. The present study was conducted in the department of otorhinolaryngology, Pt. B. D. Sharma PGIMS, Rohtak. The study was conducted on 40 patients of either sex in age group of 15-50 years having unilateral or bilateral inactive (mucosal) chronic otitis media with dry ear over a period of at least 4 weeks without use of topical or systemic antibiotics after obtaining their informed and written consent. Mean age in group I was 25.25 ± 7.27 years and in group II was 25.95 ± 9.06 years. Maximum number of patients in both groups were in the age group 15-24 years. Out of the total patients, 60% were males and 40% were females. At 6 months post operatively, 95% cases in group I had successful graft take-up compared to 85% cases in group II. However, at long term follow up for 24 months, graft success rate was statistically significant in group I. In group I, 100% graft uptake was seen in large size perforation of 4 and 5 mm along with 2 mm as compared to group II, with 100% graft uptake for only small size perforation of 2 mm. The mean hearing threshold gain was 16.50 ± 5.52 dB in group I as compared to 13.03 ± 6.44 dB in group II. Mean postoperative improvement in air bone (AB) gap of 16.50 ± 5.52 dB was seen in group I as compared to 13.07 ± 6.44 dB seen in group II. The graft take up rate was found to be better in long term with inlay cartilage- perichondrium composite graft myringoplasty technique compared to over lay technique with both the groups showing significant hearing improvement post-operatively. This high success rate for graft uptake and ease to perform under local anaesthesia makes in-lay cartilage perichondrium composite graft myringoplasty technique relatively optimal to use for office based myringoplasty. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-023-03487-w.

Isolated Frontal Sinus Mucormycosis Post Covid 19-external Approaches Revisited!

Background: Isolated frontal sinus involvement in mucormycosis is seen very infrequently. Recent technological advances including image guided navigation and angled endoscopes have shifted paradigm towards minimally invasive surgeries. Open approaches are still relevant for the disease of frontal sinus with lateral extension where effective clearance cannot be obtained if approached endoscopically. Objectives: The objective of this study was to describe the presentation and management of patients of mucormycosis with isolated frontal sinus involvement with help of external approaches. Materials and methods: The available records of the patients were retrieved and analysed. The literature, the associated contributory clinical features and management techniques were reviewed. Results: 4 patients presented with isolated frontal sinus mucor involvement. 3 out of 4 patients had history of diabetes mellitus (75%). All patients had history of covid-19 infection (100%). 3 out of 4 patients had unilateral frontal sinus involvement and were operated by Lynch Howarth approach. Mean age of presentation was 46 years with male predominance. Bicoronal approach was used in one case with bilateral involvement. Conclusion: Although conservative endoscopic surgeries are preferred nowadays for frontal sinus clearance but the extensive bony destruction with lateral extension in our series of patients with isolated frontal sinus mucormycosis warranted the need for open procedures.

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors.

BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20-57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.