A general computational design strategy for stabilizing viral class I fusion proteins.

Many pathogenic viruses rely on class I fusion proteins to fuse their viral membrane with the host cell membrane. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more stable postfusion state. Mounting evidence underscores that antibodies targeting the prefusion conformation are the most potent, making it a compelling vaccine candidate. Here, we establish a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. With this protocol, we stabilize the fusion proteins of the RSV, hMPV, and SARS-CoV-2 viruses, testing fewer than a handful of designs. The solved structures of these designed proteins from all three viruses evidence the atomic accuracy of our approach. Furthermore, the humoral response of the redesigned RSV F protein compares to that of the recently approved vaccine in a mouse model. While the parallel design of two conformations allows the identification of energetically sub-optimal positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.

Silver Nanoparticles and Its Mechanistic Insight for Chronic Wound Healing: Review on Recent Progress.

Wounds are structural and functional disruptions of skin that occur because of trauma, surgery, acute illness, or chronic disease conditions. Chronic wounds are caused by a breakdown in the finely coordinated cascade of events that occurs during healing. Wound healing is a long process that split into at least three continuous and overlapping processes: an inflammatory response, a proliferative phase, and finally the tissue remodeling. Therefore, these processes are extensively studied to develop novel therapeutics in order to achieve maximum recovery with minimum scarring. Several growth hormones and cytokines secreted at the site of lesions tightly regulates the healing processes. The traditional approach for wound management has been represented by topical treatments. Metal nanoparticles (e.g., silver, gold and zinc) are increasingly being employed in dermatology due to their favorable effects on healing, as well as in treating and preventing secondary bacterial infections. In the current review, a brief introduction on traditional would healing approach is provided, followed by focus on the potential of wound dressing therapeutic techniques functionalized with Ag-NPs.

Structural basis for ultrapotent antibody-mediated neutralization of human metapneumovirus.

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.

The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations.

Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.

A Prospective Mixed-Methods Study of Decision-Making on Surgery or Active Surveillance for Low-Risk Papillary Thyroid Cancer.

Background: Active surveillance (AS) of small, low-risk papillary thyroid cancers (PTCs) is increasingly being considered. There is limited understanding of why individuals with low-risk PTC may choose AS over traditional surgical management. Methods: We present a mixed-methods analysis of a prospective observational real-life decision-making study regarding the choice of thyroidectomy or AS for management of localized, low-risk PTCs <2 cm in maximum diameter (NCT03271892). Patients were provided standardized medical information and were interviewed after making their decision (which dictated disease management). We evaluated patients' levels of decision-self efficacy (confidence in medical decision-making ability) at the time information was presented and their level of decision satisfaction after finalizing their decision (using standardized questionnaires). We asked patients to explain the reason for their choice and qualitatively analyzed the results. Results: We enrolled 74 women and 26 men of mean age 52.4 years, with a mean PTC size of 11.0 mm (interquartile range 9.0, 14.0 mm). Seventy-one patients (71.0% [95% confidence interval 60.9-79.4%]) chose AS over surgery. Ninety-four percent (94/100) of participants independently made their own disease management choice; the rest shared the decision with their physician. Participants had a high baseline level of decision self-efficacy (mean 94.3, standard deviation 9.6 on a 100-point scale). Almost all (98%, 98/100) participants reported high decision satisfaction. Factors reported by patients as influencing their decision included the following: perceived risk of thyroidectomy or the cancer, family considerations, treatment timing in the context of life circumstances, and trust in health care providers. Conclusions: In this Canadian study, ∼7 out of 10 patients with small, low-risk PTC, who were offered the choice of AS or surgery, chose AS. Personal perceptions about cancer or thyroidectomy, contextual factors, family considerations, and trust in health care providers strongly influenced patients' disease management choices.

The Hypervariable Region of K-Ras4B Governs Molecular Recognition and Function.

The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein-protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein-protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.

The Structural Basis of the Farnesylated and Methylated KRas4B Interaction with Calmodulin.

Ca2+-calmodulin (CaM) extracts KRas4B from the plasma membrane, suggesting that KRas4B/CaM interaction plays a role in regulating Ras signaling. To gain mechanistic insight, we provide a computational model, supported by experimental structural data, of farnesylated/methylated KRas4B1-185 interacting with CaM in solution and at anionic membranes including signaling lipids. Due to multiple interaction modes, we observe diverse conformational ensembles of the KRas4B-CaM complex. A highly populated conformation reveals the catalytic domain interacting with the N-lobe and the hypervariable region (HVR) wrapping around the linker with the farnesyl docking to the extended CaM's C-lobe pocket. Alternatively, KRas4B can interact with collapsed CaM with the farnesyl penetrating CaM's center. At anionic membranes, CaM interacts with the catalytic domain with large fluctuations, drawing the HVR. Signaling lipids establishing strong salt bridges with CaM prevent membrane departure. Membrane-interacting KRas4B-CaM complex can productively recruit phosphatidylinositol 3-kinase α (PI3Kα) to the plasma membrane, serving as a coagent in activating PI3Kα/Akt signaling.

The bacterial Ras/Rap1 site-specific endopeptidase RRSP cleaves Ras through an atypical mechanism to disrupt Ras-ERK signaling.

The Ras-extracellular signal-regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor-mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.

Flexible-body motions of calmodulin and the farnesylated hypervariable region yield a high-affinity interaction enabling K-Ras4B membrane extraction.

In calmodulin (CaM)-rich environments, oncogenic KRAS plays a critical role in adenocarcinomas by promoting PI3K/Akt signaling. We previously proposed that at elevated calcium levels in cancer, CaM recruits PI3Kα to the membrane and extracts K-Ras4B from the membrane, organizing a K-Ras4B-CaM-PI3Kα ternary complex. CaM can thereby replace a missing receptor-tyrosine kinase signal to fully activate PI3Kα. Recent experimental data show that CaM selectively promotes K-Ras signaling but not of N-Ras or H-Ras. How CaM specifically targets K-Ras and how it extracts it from the membrane in KRAS-driven cancer is unclear. Obtaining detailed structural information for a CaM-K-Ras complex is still challenging. Here, using molecular dynamics simulations and fluorescence experiments, we observed that CaM preferentially binds unfolded K-Ras4B hypervariable regions (HVRs) and not α-helical HVRs. The interaction involved all three CaM domains including the central linker and both lobes. CaM specifically targeted the highly polybasic anchor region of the K-Ras4B HVR that stably wraps around CaM's acidic linker. The docking of the farnesyl group to the hydrophobic pockets located at both CaM lobes further enhanced CaM-HVR complex stability. Both CaM and K-Ras4B HVR are highly flexible molecules, suggesting that their interactions permit highly dynamic flexible-body motions. We, therefore, anticipate that the flexible-body interaction is required to extract K-Ras4B from the membrane, as conformational plasticity enables CaM to orient efficiently to the polybasic HVR anchor, which is partially diffused into the liquid-phase membrane. Our structural model of the CaM-K-Ras4B HVR association provides plausible clues to CaM's regulatory action in PI3Kα activation involving the ternary complex in cell proliferation signaling by oncogenic K-Ras.

Calmodulin and PI3K Signaling in KRAS Cancers.

Calmodulin (CaM) uniquely promotes signaling of oncogenic K-Ras; but not N-Ras or H-Ras. How CaM interacts with K-Ras and how this stimulates cell proliferation are among the most challenging questions in KRAS-driven cancers. Earlier data pointed to formation of a ternary complex consisting of K-Ras, PI3Kα and CaM. Recent data point to phosphorylated CaM binding to the SH2 domains of the p85 subunit of PI3Kα and activating it. Modeling suggests that the high affinity interaction between the phosphorylated CaM tyrosine motif and PI3Kα, can promote full PI3Kα activation by oncogenic K-Ras. Our up-to-date review discusses CaM's role in PI3K signaling at the membrane in KRAS-driven cancers. This is significant since it may help development of K-Ras-specific pharmacology.

A prospective study of magnetic resonance imaging patterns of central nervous system infections in pediatric age group and young adults and their clinico-biochemical correlation.

BACKGROUND: Infections of the central nervous system (CNS) are common and routinely encountered. Our aim was to evaluate the neuroimaging features of the various infections of the CNS so as to differentiate them from tumoral, vascular, and other entities that warrant a different line of therapy. AIMS: Our aim was to analyze the biochemical and magnetic resonance imaging (MRI) features in CNS infections. SETTINGS AND DESIGN: This was a longitudinal, prospective study over a period of 1½ years. SUBJECTS AND METHODS: We studied cerebrospinal fluid (CSF) findings and MRI patterns in 27 patients of 0-20 years age group with clinical features of CNS infections. MRI was performed on MAGNETOM Avanto 18 Channel 1.5 Tesla MR machine by Siemens India Ltd. The MRI protocol consisted of diffusion-weighted and apparent diffusion coefficient imaging, turbo spin echo T2-weighted, spin echo T1-weighted, fluid-attenuated inversion recovery (FLAIR), and gradient-echo in axial, FLAIR in coronal, and T2-weighted in sagittal plane. Contrast-enhanced T1-weighted sequence and MR spectroscopy were done whenever indicated. RESULTS AND CONCLUSIONS: We found that most of the children belong to 1-10 years age group. Fungal infections were uncommon, mean CSF adenosine deaminase values specific for tuberculosis and mean CSF glucose-lowered in pyogenic. Hemorrhagic involvement of thalamus with/without basal ganglia and brainstem involvement may indicate Japanese encephalitis or dengue encephalitis. Diffusion restriction or hemorrhage in not expected in the brainstem afflicted lesions of rabies. Congenital cytomegalovirus can cause cortical malformations. T1 hyperintensities with diffusion restriction may represent viral encephalitis. Lesions of acute disseminated encephalomyelitis (ADEM) may mimic viral encephalitis. Leptomeningeal enhancement is predominant in pyogenic meningitis. Basilar meningitis in the presence of tuberculomas is highly sensitive and specific for tuberculosis.

The higher level of complexity of K-Ras4B activation at the membrane.

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane.

The disordered hypervariable region and the folded catalytic domain of oncogenic K-Ras4B partner in phospholipid binding.

The C-terminal hypervariable region (HVR) of the splice variant KRAS4B is disordered. Classically, the role of the post-translationally-modified HVR is to navigate Ras in the cell and to anchor it in localized plasma membrane regions. Here, we propose additional regulatory roles, including auto-inhibition by shielding the effector binding site in the GDP-bound state and release upon GTP binding and in the presence of certain oncogenic mutations. The released HVR can interact with calmodulin. We show that oncogenic mutations (G12V/G12D) modulate the HVR-phospholipid binding specificity, resulting in preferential interactions with phosphatidic acid. The shifts in the conformational preferences and binding specificity in the disordered state exemplify the critical role of the unstructured tail of K-Ras4B in cancer.

High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site.

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding.

Tuberous sclerosis complex presenting as pulmonary lymphangioleiomyomatosis--a clinicoradiological diagnosis.

Tuberous sclerosis complex (TSC) manifests predominantly as a neurocutaneous disorder. Lymphangioleiomyomatosis (LAM) is a rare pulmonary manifestation of TSC. Imaging evaluation plays an important role in the assessment of patients with tuberous sclerosis complex. In newly diagnosed patients, it helps not only to confirm the diagnosis of TSC, but also helps in identifying clinically significant complications. We describe the radiological findings in lungs and other organs in a middle aged female with TSC.

Conserved water mediated H-bonding dynamics of Ser117 and Thr119 residues in human transthyretin-thyroxin complexation: inhibitor modeling study through docking and molecular dynamics simulation.

Transthyretin (TTR) is a protein whose aggregation and deposition causes amyloid diseases in human beings. Amyloid fibril formation is prevented by binding of thyroxin (T4) or its analogs to TTR. The MD simulation study of several solvated X-ray structures of apo and holo TTR has indicated the role of a conserved water molecule and its interaction with T4 binding residues Ser117 and Thr119. Geometrical and electronic consequences of those interactions have been exploited to design a series of thyroxin analogs (Mod1-4) by modifying 5' or 3' or both the iodine atoms of thyroxin. Binding energy of the designed ligands has been calculated by docking the molecules in tetrameric structure of the protein. Theoretically investigated pharmacological parameters along with the binding energy data indicate the potentiality of 3',5'-diacetyl-3,5-dichloro-l-thyronine (Mod4) to act as a better inhibitor for TTR-related amyloid diseases.

Xanthine oxidoreductase: a journey from purine metabolism to cardiovascular excitation-contraction coupling.

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective "housekeeping enzyme". It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.

Rounding of the inferior rectus muscle as a predictor of enophthalmos in orbital floor fractures.

In spite of established indications for early operative repair of orbital floor fractures 7-10% of patients treated nonoperatively develop enophthalmos. Clearly further indications for repair are required to prevent these post-injury complications. Rounding of the inferior rectus muscle on coronal computerized tomography (CT) scan results from a loss of soft tissue and bony support and may therefore be predictive of late enophthalmos.A four-year institutional review was conducted to identify patients with orbital floor fractures that had been treated nonoperatively. Patients were recruited for late clinical follow-up (mean 30 months) where clinically significant enophthalmos and diplopia were measured. Clinical results were correlated with measurements of the height-to-width ratio of the inferior rectus muscle on CT scans by a blinded examiner. Eighteen of 78 patients were available for late follow-up. Sixteen patients had no enophthalmos whereas 2 patients had enophthalmos. The inferior rectus height-to-width ratios measured in the unaffected orbits were statistically similar between the two groups. There was a significantly increased height-to-width ratio exceeding 1.00 in the affected orbit when the enophthalmos group was compared to the no enophthalmos group.A height-to-width ratio of the inferior rectus muscle on coronal CT scan of greater than or equal to 1.00 is predictive of late enophthalmos.