The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway.

(L597V)BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline (L597V)BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of (L597V)Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined (L597V)Braf with (G12D)Kras and found that (L597V)Braf modified (G12D)Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity (V600E)Braf mutant. Mek/Erk activation levels were comparable with those driven by (V600E)Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by (V600E)Braf than (G12D)Kras. However, unlike (V600E)Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, (L597V)Braf epistatically modifies the transforming effects of driver oncogenes.

Existing and emerging applications for the neuromodulation of nerve activity through targeted delivery of electric stimuli.

The effective treatment of many diseases requires the use of multiple treatment strategies among which neuromodulation is playing an increasingly important role. Neuromodulation devices that act to normalize or modulate nerve activity through the targeted delivery of electrical stimuli will be the focus of this review. These devices encompass deep brain stimulators, vagus nerve stimulators, spinal cord simulators and sacral nerve stimulators. Already neuromodulation has proven successful in the treatment of a broad range of conditions from Parkinson's disease to chronic pain and urinary incontinence. Many of these approaches seek to exploit the activities of the autonomic nervous system, which influences organ function through the release of neurotransmitters and associated signalling cascades. This review will outline existing and emerging applications for each of these neuromodulation devices, proposed mechanisms of action and clinical studies evaluating both their safety and therapeutic efficacy.

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation.

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for beta-integrin tails, F0 and F1 are also required for activation of beta1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of beta1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.

A Narrative Overview of Coronavirus Infection: Clinical Signs and Symptoms, Viral Entry and Replication, Treatment Modalities, and Management.

The global pandemic known as coronavirus disease (COVID-19) is causing morbidity and mortality on a daily basis. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV--2) virus has been around since December 2019 and has infected a high number of patients due to its idiopathic pathophysiology and rapid transmission. COVID-19 is now deemed a newly identified "syndrome" condition since it causes a variety of unpleasant symptoms and systemic side effects following the pandemic. Simultaneously, it always becomes potentially hazardous when new variants develop during evolution. Its random viral etiology prevents accurate and suitable therapy. Despite the fact that multiple preclinical and research studies have been conducted to combat this lethal virus, and various therapeutic targets have been identified, the precise course of therapy remains uncertain. However, just a few drugs have shown efficacy in treating this viral infection in its early stages. Currently, several medicines and vaccinations have been licensed following clinical trial research, and many countries are competing to find the most potent and effective immunizations against this highly transmissible illness. For this narrative review, we used PubMed, Google Scholar, and Scopus to obtain epidemiological data, pre-clinical and clinical trial outcomes, and recent therapeutic alternatives for treating COVID-19 viral infection. In this study, we discussed the disease's origin, etiology, transmission, current advances in clinical diagnostic technologies, different new therapeutic targets, pathophysiology, and future therapy options for this devastating virus. Finally, this review delves further into the hype surrounding the SARS-CoV-2 illness, as well as present and potential COVID-19 therapies.

Structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation.

Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1-R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length talin at a resolution of approximately 2.5nm using EM reconstruction of full-length talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two talin rods form a donut-shaped structure, with the two talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that talin must unravel before it can support integrin activation and engage the actin cytoskeleton.

Regulation of MEK/ERK pathway output by subcellular localization of B-Raf.

The strength and duration of intracellular signalling pathway activation is a key determinant of the biological outcome of cells in response to extracellular cues. This has been particularly elucidated for the Ras/Raf/MEK [mitogen-activated growth factor/ERK (extracellular-signal-regulated kinase) kinase]/ERK signalling pathway with a number of studies in fibroblasts showing that sustained ERK signalling is a requirement for S-phase entry, whereas transient ERK signalling does not have this capability. A major unanswered question, however, is how a cell can sustain ERK activation, particularly when ERK-specific phosphatases are transcriptionally up-regulated by the pathway itself. A major point of ERK regulation is at the level of Raf, and, to sustain ERK activation in the presence of ERK phosphatases, sustained Raf activation is a requirement. Three Raf proteins exist in mammals, and the activity of all three is induced following growth factor stimulation of cells, but only B-Raf activity is maintained at later time points. This observation points to B-Raf as a regulator of sustained ERK activation. In the present review, we consider evidence for a link between B-Raf and sustained ERK activation, focusing on a potential role for the subcellular localization of B-Raf in this key physiological event.

Vagus Nerve Stimulation as a Potential Therapy in Early Alzheimer's Disease: A Review.

Cognitive dysfunction in Alzheimer's disease (AD) is caused by disturbances in neuronal circuits of the brain underpinned by synapse loss, neuronal dysfunction and neuronal death. Amyloid beta and tau protein cause these pathological changes and enhance neuroinflammation, which in turn modifies disease progression and severity. Vagal nerve stimulation (VNS), via activation of the locus coeruleus (LC), results in the release of catecholamines in the hippocampus and neocortex, which can enhance synaptic plasticity and reduce inflammatory signalling. Vagal nerve stimulation has shown promise to enhance cognitive ability in animal models. Research in rodents has shown that VNS can have positive effects on basal synaptic function and synaptic plasticity, tune inflammatory signalling, and limit the accumulation of amyloid plaques. Research in humans with invasive and non-invasive VNS devices has shown promise for the modulation of cognition. However, the direct stimulation of the vagus nerve afforded with the invasive procedure carries surgical risks. In contrast, non-invasive VNS has the potential to be a broadly available therapy to manage cognitive symptoms in early AD, however, the magnitude and specificity of its effects remains to be elucidated, and the non-inferiority of the effects of non-invasive VNS as compared with invasive VNS still needs to be established. Ongoing clinical trials with healthy individuals and patients with early AD will provide valuable information to clarify the potential benefits of non-invasive VNS in cognition and AD. Whether invasive or non-invasive VNS can produce a significant improvement on memory function and whether its effects can modify the progression of AD will require further investigation.

Central region of talin has a unique fold that binds vinculin and actin.

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359-1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel ß-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.

Fibroblast-Derived STC-1 Modulates Tumor-Associated Macrophages and Lung Adenocarcinoma Development.

The tumor microenvironment (TME) consists of different cell types, including tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs). How these cells interact and contribute to lung carcinogenesis remains elusive. Using G12DKRAS- and V600EBRAF-driven mouse lung models, we identify the pleiotropic glycoprotein stanniocalcin-1 (STC1) as a regulator of TAM-TAF interactions. STC1 is secreted by TAFs and suppresses TAM differentiation, at least in part, by sequestering the binding of GRP94, an autocrine macrophage-differentiation-inducing factor, to its cognate scavenger receptors. The accumulation of mature TAMs in the Stc1-deficient lung leads to enhanced secretion of TGF-ß1 and, thus, TAF accumulation in the TME. Consistent with the mouse data, in human lung adenocarcinoma, STC1 expression is restricted to myofibroblasts, and a significant increase of naive macrophages is detected in STC1-high compared with STC1-low cases. This work increases our understanding of lung adenocarcinoma development and suggests new approaches for therapeutic targeting of the TME.

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.

A vinculin binding domain from the talin rod unfolds to form a complex with the vinculin head.

The cytoskeletal protein talin plays a key role in activating integrins and in coupling them to the actin cytoskeleton. Its N-terminal globular head, which binds beta integrins, is linked to an extended rod having a C-terminal actin binding site and several vinculin binding sites (VBSs). The NMR structure of residues 755-889 of the rod (containing a VBS) is shown to be an amphipathic four-helix bundle with a left-handed topology. A talin peptide corresponding to the VBS binds the vinculin head; the X-ray crystallographic structure of this complex shows that the residues which interact with vinculin are buried in the hydrophobic core of the talin fragment. NMR shows that the interaction involves a major structural change in the talin fragment, including unfolding of one of its helices, making the VBS accessible to vinculin. Interestingly, the talin 755-889 fragment binds more than one vinculin head molecule, suggesting that the talin rod may contain additional as yet unrecognized VBSs.

Regulation of BRAF protein stability by a negative feedback loop involving the MEK-ERK pathway but not the FBXW7 tumour suppressor.

The (V600E)BRAF oncogenic mutation is detected in a wide range of human cancers and induces hyperactivation of the downstream MEK-ERK signalling cascade. Although output of the BRAF-MEK-ERK pathway is regulated by feed-forward RAF activity, feedback control also plays an important role. One such feedback pathway has been identified in Caenorhabditis elegans and involves ERK-mediated phosphorylation of BRAF within a CDC4 phosphodegron (CPD), targeting BRAF for degradation via CDC4 (also known as FBXW7), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex. Here we investigate this pathway in mammalian cells. Short-term expression of autochthonous (V600E)BRAF in mouse embryonic fibroblasts (MEFs) leads to down-regulation of BRAF protein levels in a proteasome-dependent manner and (V600E)BRAF has a reduced half-life compared to (WT)BRAF in HEK293(T) cells. These effects were reversed by treatment with the MEK inhibitor PD184352. We have identified the equivalent CPD at residues 400-405 in human BRAF and have found that mutation of ERK phosphorylation sites at residues T401 and S405 in (V600E)BRAF increases the half-life of the protein. While BRAF and FBXW7 co-immunoprecipitated, the overexpression of FBXW7 did not influence the half-life of either (WT)BRAF or (V600E)BRAF. Furthermore, disruption of the substrate-binding site of mouse FBXW7 using the R482Q mutation did not affect the interaction with BRAF and the expression levels of (WT)BRAF and (V600E)BRAF were not altered in MEFs derived from mice with the homozygous knockin (R482Q)FBXW7 mutation. Overall these data confirm the existence of a negative feedback pathway by which BRAF protein stability is regulated by ERK. However, unlike the situation in C. elegans, FBXW7 does not play a unique role in mediating subsequent BRAF degradation.

Characterization of an actin-binding site within the talin FERM domain.

Talin is a large cytoskeletal protein that couples integrins to F-actin. Three actin-binding sites (ABS1-3) have been reported: one in the N-terminal head, and two in the C-terminal rod domain. Although the C-terminal ABS3 has been partially characterized, the presence and properties of ABS1 within the talin head are less well defined. We show here that the talin head binds F-actin in vitro and in vivo at a specific site within the actin filament. Thus, purified talin head liberated from gizzard talin by calpain cleavage cosediments with F-actin in a low salt buffer at pH 6.4 (conditions that are optimal for binding intact talin), and using recombinant polypeptides, we have mapped ABS1 to the FERM domain within the talin head. Both the F2 and F3 FERM subdomains contribute to binding, and EGFP-tagged FERM subdomains colocalize with actin stress fibers when expressed in COS cells. High-resolution electron microscopy of actin filaments decorated with F2F3 localizes binding to a site that is distinct from that recognized by members of the calponin-homology superfamily. Finally, we show that the FERM domain can couple F-actin to PIPkin, and by inference to integrins, since they bind to the same pocket in the F3 subdomain. This suggests that the talin FERM domain functions as a linker between PIPkin or integrins and F-actin at sites of cell-matrix adhesions.

The effect of load obliquity on the strength of locking and nonlocking constructs in synthetic osteoporotic bone.

The biomechanical performance of internal fracture fixation depends on several factors. One measure of performance is the strength of the construct. The objective of this biomechanical study was to identify the effect of load obliquity on the strength of locking and nonlocking plate and screw constructs. For this study, plates and screws were fixed to synthetic osteoporotic bone that had a 1 mm thick synthetic cortical shell. An 8-hole, 3.5 mm thick hybrid plate was fixed with either two 3.5 mm major diameter locking screws or two 4.0 mm major diameter cancellous screws. Forces were applied at 0, 45, and 90 degrees to the plate normal. Eight specimens were loaded to failure for each group. When loads were applied normal to the plate, the nonlocking construct failed initially at higher loads (123.2 ± 13.2 N) than the locking construct (108.7 ± 7.6 N, P = 0.020). For oblique loads, the locking construct failed at higher mean loads but the difference of means was not statistically significant (167.7 ± 14.9 N compared to 154.2 ± 9.4 N, P = 0.052). For loads parallel to the plate, the locking construct was much stronger than the nonlocking construct (1591 ± 227 N compared to 913 ± 237 N, P < 0.001). Stiffness and Energy outcomes are also compared.

The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours.

The tumour microenvironment is known to play an integral role in facilitating cancer progression at advanced stages, but its function in some pre-cancerous lesions remains elusive. We have used the (V600) (E)BRAF-driven mouse lung model that develop premalignant lesions to understand stroma-tumour interactions during pre-cancerous development. In this model, we have found that immature macrophage-lineage cells (IMCs) producing PDGFA, TGFß and CC chemokines are recruited to the stroma of premalignant lung adenomas through CC chemokine receptor 1 (CCR1)-dependent mechanisms. Stromal IMCs promote proliferation and transcriptional alterations suggestive of epithelial-mesenchymal transition in isolated premalignant lung tumour cells ex vivo, and are required for the maintenance of early-stage lung tumours in vivo. Furthermore, we have found that IMC recruitment to the microenvironment is restrained by the cholesterol-binding protein, Niemann-Pick type C2 (NPC2). Studies on isolated cells ex vivo confirm that NPC2 is secreted from tumour cells and is taken up by IMCs wherein it suppresses secretion of the CCR1 ligand CC chemokine 6 (CCL6), at least in part by facilitating its lysosomal degradation. Together, these findings show that NPC2 secreted by premalignant lung tumours suppresses IMC recruitment to the microenvironment in a paracrine manner, thus identifying a novel target for the development of chemopreventive strategies in lung cancer.

Development of a sensitive high-performance thin-layer chromatography method for estimation of ranitidine in urine and its application for bioequivalence decision for ranitidine tablet formulations.

A sensitive and simple HPTLC method was developed for estimation of ranitidine in human urine. The drug was extracted from urine after basification using dichloromethane. Dichloromethane extract was spotted on silica gel 60 F254 TLC plate and was developed in a mixture of ethyl acetate-methanol-ammonia (35:10:5 v/v) as the mobile phase and scanned at 320 nm. The RF value obtained for the drug was 0.67 +/- 0.03. The method was validated in terms of linearity (50-400 ng/spot), precision and accuracy. The average recovery of ranitidine from urine was 89.35%. The proposed method was applied to evaluate bioequivalence of two marketed ranitidine tablet formulations (150 mg, Formulation I and Formulation 2) using a crossover design by comparing urinary excretion data for unchanged ranitidine in six healthy volunteers. Various pharmacokinetic parameters like peak excretion rate [(dAU/dt)max], time for peak excretion rate (tmax), AUC0-24, AUC0-infinity, cumulative amount excreted were calculated for both formulations and subjected to statistical analysis. The relative bioavailability of Formulation 2 with respect to Formulation 1 was 93.76 and 95.31% on the basis of AUC0-24 and cumulative amount excreted, respectively. Statistical comparison of various pharmacokinetic parameters indicated that the two ranitidine tablet formulations are bioequivalent.

Combined Interatrial and Intrapulmonary Shunting in Orthodeoxia Detected by Transesophageal Echocardiography.

Contrast transesophageal echocardiography was found useful in diagnosing combined interatrial and intrapulmonary right-to-left shunts in a patient presenting with orthodeoxia. This was done by separately examining the pulmonary veins and the interatrial septum during intravenous normal saline injections.

Evaluation of polyherbal formulation (SJT-HT-03) for antihypertensive activity in albino rats.

BACKGROUND: Hypertension is an incurable pathological condition and lifelong therapy is required. Long term use of conventional synthetic anti-hypertensive drugs is associated with a spectrum of toxic effects. However, therapeutic interventions using herbal drugs for hypertension have gained considerable attention worldwide. AIM: To evaluate the anti-hypertensive activity of polyherbal formulation (SJT-HT-03). MATERIALS AND METHODS: The polyherbal formulation (SJT-HT-03) comprises of leaves of Aegle marmelos L., fruits of Benincasa hispida Thunb., Garcinia indica Thouars, and flowers of Musa paradiasica L., Rosa indica L., Hibiscus rosa sinensis L. Selected plants as mentioned above were collected, dried and extracted with different solvents. Formulation SJT-HT-03 (250 mg/kg, p.o.), was evaluated using two kidney one clip (2K1C) model and deoxycorticosterone acetate (DOCA)-salt-induced hypertension model using the enalapril (10 mg/kg, p.o.) and hydrochlorothiazide (5 mg/kg, p.o.) as a reference standard drug in respective models. RESULTS: SJT-HT-03 significantly reduced (P < 0.001, one-way analysis of variance followed by Turkey's multiple comparison tests) systolic as well as diastolic blood pressure (BP) in 2K1C and DOCA-salt model. Further, SJT-HT-03 has shown a significant reduction (P < 0.01) in angiotensin converting enzyme (ACE) activity in serum, clipped kidney as well as in lungs in 2K1C model, whereas significant reduction (P < 0.05) in serum Na(+) and increase in serum K(+) level in DOCA model. CONCLUSION: Polyherbal formulation SJT-HT-03 possess significant anti-hypertensive activity by producing direct depressant effect on heart, inhibition of ACE, aldosterone antagonistic as well as diuretic effect and thereby act on multiple targets to achieve optimal effect.