Recent Advances On Direct Formylation Reactions.

Aldehydes serve as the key functional group in organic synthesis and are valuable intermediates. The various advanced methods of direct formylation reactions have been reviewed in this article. Overcoming the drawbacks of the traditional methods of formylation, newer methods involving homo and heterogenous catalysts, one pot reactions, solvent free techniques are elaborated, which can be performed under mild conditions and using inexpensive resources.

Integration of CRISPR/Cas9 with artificial intelligence for improved cancer therapeutics.

Gene editing has great potential in treating diseases caused by well-characterized molecular alterations. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene-editing tools has substantially improved the precision and efficiency of gene editing. The CRISPR/Cas9 system offers several advantages over the existing gene-editing approaches, such as its ability to target practically any genomic sequence, enabling the rapid development and deployment of novel CRISPR-mediated knock-out/knock-in methods. CRISPR/Cas9 has been widely used to develop cancer models, validate essential genes as druggable targets, study drug-resistance mechanisms, explore gene non-coding areas, and develop biomarkers. CRISPR gene editing can create more-effective chimeric antigen receptor (CAR)-T cells that are durable, cost-effective, and more readily available. However, further research is needed to define the CRISPR/Cas9 system's pros and cons, establish best practices, and determine social and ethical implications. This review summarizes recent CRISPR/Cas9 developments, particularly in cancer research and immunotherapy, and the potential of CRISPR/Cas9-based screening in developing cancer precision medicine and engineering models for targeted cancer therapy, highlighting the existing challenges and future directions. Lastly, we highlight the role of artificial intelligence in refining the CRISPR system's on-target and off-target effects, a critical factor for the broader application in cancer therapeutics.

Prognostic Nutritional Index Prior to Rectal Cancer Resection Predicts Overall Survival.

Prognostic nutritional index (PNI) correlates with postoperative complications and survival in colorectal cancers. Separate studies for rectal cancers are not available where the majority have preoperative radiation, operated by minimally invasive approaches and have diverting ostomies.Consecutive rectal resections between October 2014 and December 2017 from a single center were included. PNI was calculated as 10 x (serum Albumin) + 0.005 x TLC (per mm3) before operation. Multivariate cox regression was used with overall survival (OS) as the dependent variable. Interaction terms of PNI with neoadjuvant therapy, surgical approach and postoperative complications were used to assess specific subgroups.Three-hundred forty elective rectal resections were included with a mean PNI of 46.711 (SD - 6.692), and a median follow up of 44 mo. In multivariable regression, PNI predicted OS (HR - 0.943; p-0.001). Interaction of PNI with preoperative radiation or surgical approach (open, laparoscopic, or robotic) did not change its influence on survival. PNI predicted survival with similar hazard even in patients without major postoperative complicationsDespite routine diversion after rectal resections, PNI predicted OS with an absolute survival benefit of 1.2% at 3-year for every unit increase in PNI irrespective of preoperative therapy or surgical approach.

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating.

In K(+) channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K(+) channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K(+) channels.

Targeting mitochondria as a potential therapeutic strategy against chemoresistance in cancer.

The importance of mitochondria is not only limited to energy generation but also in several physical and chemical processes critical for cell survival. Mitochondria play an essential role in cellular apoptosis, calcium ion transport and cellular metabolism. Mutation in the nuclear and mitochondrial genes, altered oncogenes/tumor suppressor genes, and deregulated signalling for cell viability are major reasons for cancer progression and chemoresistance. The development of drug resistance in cancer patients is a major challenge in cancer treatment as the resistant cells are often more aggressive. The drug resistant cells of numerous cancer types exhibit the deregulation of mitochondrial function. The increased biogenesis of mitochondria and its dynamic alteration contribute to developing resistance. Further, a small subpopulation of cancer stem cells in the heterogeneous tumor is primarily responsible for chemoresistance and has an attribute of mitochondrial dysfunction. This review highlights the critical role of mitochondrial dysfunction in chemoresistance in cancer cells through the processes of apoptosis, autophagy/mitophagy, and cancer stemness. Mitochondria-targeted therapeutic strategies might help reduce cancer progression and chemoresistance induced by various cancer drugs.

CCL2 and CCL3 are essential mediators of pelvic pain in experimental autoimmune prostatitis.

Experimental autoimmune prostatitis (EAP) is a murine model of chronic prostatitis/chronic pelvic pain syndrome (CPPS) in men, a syndrome characterized by chronic pelvic pain. We have demonstrated that chemokine ligands CCL2 and CCL3 are biomarkers that correlate with pelvic pain symptoms. We postulated that CCL2 and CCL3 play a functional role in CPPS and therefore examined their expression in EAP. Upon examination of the prostate 5 days after induction of EAP, CCL2 mRNA was elevated 2- to 3-fold, CCL8 by 15-fold, CCL12 by 12- to 13-fold, and CXCL9 by 2- to 4-fold compared with control mice. At 10 days the major chemokines were CXCL13 and CXCL2; at 20 days CCL2 (1- to 2-fold), CCL3 (2- to 3-fold) and CCL11 (2- to 3-fold); and at 30 days, CCL12 (20- to 35-fold) and smaller increases in CCL2, CCL3, and XCL1. Chemokine elevations were accompanied by increases in mast cells and B cells at 5 days, monocytes and neutrophils at day 10, CD4+ T cells at day 20, and CD4+ and CD8+ T cells at day 30. Anti-CCL2 and anti-CCL3 neutralizing antibodies administered at EAP onset attenuated pelvic pain development, but only anti-CCL2 antibodies were effective therapeutically. CCL2- and its cognate receptor CCR2-deficient mice were completely protected from development of pain symptoms but assumed susceptibility after reconstitution with wild-type bone marrow. CCL3-deficient mice showed resistance to the maintenance of pelvic pain while CCR5-deficient mice did not show any lessening of pelvic pain severity. These results suggest that the CCL2-CCR2 axis and CCL3 are important mediators of chronic pelvic pain in EAP.

Matrix mechanics regulates epithelial defence against cancer by tuning dynamic localization of filamin.

In epithelia, normal cells recognize and extrude out newly emerged transformed cells by competition. This process is the most fundamental epithelial defence against cancer, whose occasional failure promotes oncogenesis. However, little is known about what factors determine the success or failure of this defence. Here we report that mechanical stiffening of extracellular matrix attenuates the epithelial defence against HRasV12-transformed cells. Using photoconversion labelling, protein tracking, and loss-of-function mutations, we attribute this attenuation to stiffening-induced perinuclear sequestration of a cytoskeletal protein, filamin. On soft matrix mimicking healthy epithelium, filamin exists as a dynamically single population, which moves to the normal cell-transformed cell interface to initiate the extrusion of transformed cells. However, on stiff matrix mimicking fibrotic epithelium, filamin redistributes into two dynamically distinct populations, including a new perinuclear pool that cannot move to the cell-cell interface. A matrix stiffness-dependent differential between filamin-Cdc42 and filamin-perinuclear cytoskeleton interaction controls this distinctive filamin localization and hence, determines the success or failure of epithelial defence on soft versus stiff matrix. Together, our study reveals how pathological matrix stiffening leads to a failed epithelial defence at the initial stage of oncogenesis.

Wireless COVID-19 Telehealth: Leukocytes Encryption Guided by Amino Acid Matrix.

In this era of wireless COVID-19 telehealth, visiting hospital for regular follow-ups could invite coronavirus in someone's body. Opting for proactive E-health services is the best thing. It helps the remote patients to share their confidential data through secured encryption. Telehealth services are emerging element in these proactive medical sciences. It helps the remote patients to share their confidential data through secured transmission. In this paper, amino acid guided matrix encoding scheme has been proposed. White blood cell count or Leukocute count is a dominant indicator of patients' health condition, even amid COVID-19. An abnormal growth in leukocyte count is mainly caused due to an infection, cancer, or any other severe symptoms. It initiates internal haematological inflammations, cardiovascular diseases, Type II diabetes, etc. Therefore, tracking leukocyte count may for disease diagnosis and further treatments. The leukocyte count is generally done in different pathologies, and the data evaluation needs the expertise of a pathologist. In this paper, a technique involving security measures to transmit the result of the histological test with the help of cryptography has been proposed. The data to be transferred to the concerned physician for further diagnosis with the help of proposed way of encryption using amino acids, which ensures no data loss, no data modification, no data theft in the middle of transmission. The proposed encryption method using the amino acid codes has produced results showing satisfactory performances such as p-values found to be 7.215544e-04 and 8.48904e-03 for the key stream and cipher key matrix monobit test respectively, and 8.10245e-04 and 8.10245e-04 for the key stream and cipher key matrix frequency test respectively. It may be used as a transmission module in any wireless COVID-19 Telehealth Systems.

Membrane localization and dynamics of Nile Red: effect of cholesterol.

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.

Dynamics and heterogeneity of bovine hippocampal membranes: role of cholesterol and proteins.

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.

Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching.

Lateral diffusion of membrane constituents plays an important role in membrane organization and represents a central theme in current models describing the structure and function of biological membranes. Fluorescence recovery after photobleaching (FRAP) is a widely used approach that provides information regarding dynamic properties and spatial distribution of membrane constituents. On the basis of the unique concentration-dependent fluorescence emission properties of a fluorescently labeled cholesterol analogue modified at the tail region, 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (25-NBD-cholesterol), we have previously shown that it exhibits local organization even at very low concentrations in membranes. In this paper, we address aspects regarding the molecular size and dynamics of such an organized assembly of 25-NBD-cholesterol by monitoring its lateral diffusion characteristics using FRAP. To obtain a comprehensive understanding of the organization and dynamics of 25-NBD-cholesterol in the membrane, we compare its diffusion properties to that of a fluorescent phospholipid analogue 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-(1,3-benzoxadiazol-4-yl)) (NBD-PE). Our results indicate significant differences in the membrane dynamics of these NBD-labeled lipids. Importantly, on the basis of a novel wavelength-selective FRAP approach, our results show that the organization of 25-NBD-cholesterol is heterogeneous, with the presence of fast- and slow-diffusing species which could correspond to predominant populations of monomers and dimers of 25-NBD-cholesterol. The potential application of the wavelength-selective FRAP approach to monitor the organization and dynamics of molecules in membranes therefore represents an exciting possibility.

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization.

Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.

Monitoring cholesterol organization in membranes at low concentrations utilizing the wavelength-selective fluorescence approach.

We previously showed using a fluorescent analogue of cholesterol (NBD-cholesterol, or 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol), that cholesterol may exhibit local organization at low concentrations in membranes by the formation of transbilayer tail-to-tail dimers of cholesterol (Rukmini, R., Rawat, S.S., Biswas, S.C., Chattopadhyay, A., 2001. Biophys. J. 81, 2122-2134). In this report, we have monitored the microenvironmental features of cholesterol monomers and dimers utilizing wavelength-selective fluorescence spectroscopy. Our results utilizing red edge excitation shift (REES) and wavelength-dependent change in fluorescence anisotropy show that the microenvironment around the NBD moieties in the dimer form is more rigid possibly due to steric constraints imposed by the dimer conformation. These results provide new information and are relevant in understanding the organization of cholesterol in membranes at low concentrations.

Organization and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: a fluorescence approach.

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. NBD-labeled lipids have previously been used to monitor the organization and dynamics of molecular assemblies such as membranes, micelles and reverse micelles utilizing the wavelength-selective fluorescence approach. In this paper, we have characterized the organization and dynamics of various NBD-labeled lipids using red edge excitation shift (REES) and other fluorescence approaches which include analysis of membrane penetration depths of the NBD group using the parallax method. We show here that the environment and location experienced by the NBD group of the NBD-labeled lipids could depend on the ionization state of the lipid. This could have potentially important implications in future studies involving NBD-labeled lipids as tracers in a cellular context.

Th1-Th17 cells contribute to the development of uropathogenic Escherichia coli-induced chronic pelvic pain.

The etiology of chronic prostatitis/chronic pelvic pain syndrome in men is unknown but may involve microbes and autoimmune mechanisms. We developed an infection model of chronic pelvic pain in NOD/ShiLtJ (NOD) mice with a clinical Escherichia coli isolate (CP-1) from a patient with chronic pelvic pain. We investigated pain mechanisms in NOD mice and compared it to C57BL/6 (B6) mice, a strain resistant to CP-1-induced pain. Adoptive transfer of CD4+ T cells, but not serum, from CP-1-infected NOD mice was sufficient to induce chronic pelvic pain. CD4+ T cells in CP-1-infected NOD mice expressed IFN-γ and IL-17A but not IL-4, consistent with a Th1/Th17 immune signature. Adoptive transfer of ex-vivo expanded IFN-γ or IL-17A-expressing cells was sufficient to induce pelvic pain in naïve NOD recipients. Pelvic pain was not abolished in NOD-IFN-γ-KO mice but was associated with an enhanced IL-17A immune response to CP1 infection. These findings demonstrate a novel role for Th1 and Th17-mediated adaptive immune mechanisms in chronic pelvic pain.

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes.

Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.

Influence of ester and ether linkage in phospholipids on the environment and dynamics of the membrane interface: a wavelength-selective fluorescence approach.

We have monitored the environment and dynamics of the membrane interface formed by the ester-linked phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the ether-linked phospholipid 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) utilizing the wavelength-selective fluorescence approach and using the fluorescent membrane probe 2-(9-anthroyloxy)stearic acid (2-AS). This interfacially localized probe offers a number of advantages over those which lack a fixed location in the membrane. When incorporated in membranes formed by DPPC and DHPC, 2-AS exhibits red edge excitation shift (REES) of 14 and 8 nm, respectively. This implies that the rate of solvent reorientation, as sensed by the interfacial anthroyloxy probe, in ester-linked DPPC membranes is slow compared to the rate of solvent reorientation in ether-linked DHPC membranes. In addition, the fluorescence polarization values of 2-AS are found to be higher in DHPC membranes than in DPPC membranes. This is further supported by wavelength-dependent changes in fluorescence polarization and lifetime. Taken together, these results are useful in understanding the role of interfacial chemistry on membrane physical properties.