Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent.

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.

Lattice defects induced by microtubule-stabilizing agents exert a long-range effect on microtubule growth by promoting catastrophes.

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.

Current standards of surgical management of gastric cancer: an appraisal.

PURPOSE: Gastric cancer (GC) is the fifth most common malignancy worldwide and portends a grim prognosis due to a lack of appreciable improvement in 5-year survival. We aimed to analyze the available literature and summarize the current standards of surgical care for curative and palliative intent treatment of GC. METHODS: We conducted a systematic search on the PubMed database for studies on the management of GC. RESULTS: Endoscopic resection is an acceptable treatment option for T1a tumors. The role of optimal resection margin for GC remains unclear. D2 lymph node dissection remains the standard of care with splenectomy needed selectively for splenic hilum involvement. A distal pancreatic resection should be avoided. The advantage of bursectomy and omentectomy in GC surgery is not clear. Multi-visceral resection may be considered for locally advanced GC in carefully selected patients. Minimally invasive approaches are non-inferior to open surgery. Surgery should be abandoned prior even in metastatic GC within the frame of multimodal therapy approach. CONCLUSION: Various trials have conclusively shown improved patient outcomes when well-established surgical standards are followed.

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

Photoswitchable Epothilone-Based Microtubule Stabilisers Allow GFP-Imaging-Compatible, Optical Control over the Microtubule Cytoskeleton.

Optical methods to modulate microtubule dynamics show promise for reaching the micron- and millisecond-scale resolution needed to decrypt the roles of the cytoskeleton in biology. However, optical microtubule stabilisers are under-developed. We introduce "STEpos" as GFP-orthogonal, light-responsive epothilone-based microtubule stabilisers. They use a novel styrylthiazole photoswitch in a design to modulate hydrogen-bonding and steric effects that control epothilone potency. STEpos photocontrol microtubule dynamics and cell division with micron- and second-scale spatiotemporal precision. They substantially improve potency, solubility, and ease-of-use compared to previous optical microtubule stabilisers, and the structure-photoswitching-activity relationship insights in this work will guide future optimisations. The STEpo reagents can contribute greatly to high-precision research in cytoskeleton biophysics, cargo transport, cell motility, cell division, development, and neuroscience.

Taxanes convert regions of perturbed microtubule growth into rescue sites.

Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.

Inhibition of polo-like kinase 1 suppresses microtubule dynamics in MCF-7 cells.

Polo-like kinase 1 (Plk1) is a mitotic serine/threonine kinase implicated in spindle formation and cytokinesis in mammalian cells. Here, purified Plk1 was found to bind to reconstituted microtubules in vitro. Further, Plk1 was found to co-localize with interphase microtubules in MCF-7 cells and to co-immunoprecipitate with polymerized tubulin. The binding of Plk1 to interphase microtubules appeared to increase with an increase in the level of tubulin acetylation in MCF-7 cells. Interestingly, Plk1 inhibitor III, an inhibitor of Plk1 kinase activity, treatment increased the association of Plk1 with the interphase microtubules in MCF-7 cells. Therefore, the effect of inhibition of Plk1 kinase activity on the dynamic instability of microtubules was determined by time-lapse imaging in MCF-7 cells. Plk1 inhibitor III dampened the dynamic instability of microtubules. For example, Plk1 inhibitor III (3 µM) reduced the rate and extent of the growing phase by 28 and 48%, respectively, and inhibited the dynamicity of microtubules by 53% as compared to the microtubules in control MCF-7 cells. Plk1 inhibitor III treatment also increased the level of acetylated microtubules, indicating that it stabilizes microtubules. The findings indicated that Plk1 interacts with microtubules and Plk1 may have a role in the regulation of microtubule dynamics.

Anatomical variation of the Psoas Valley: a scoping review.

BACKGROUND: This scoping review aimed to investigate the literature on the anatomy of the psoas valley, an anterior depression on the acetabular rim, and propose a unified definition of the anatomical structure, describe its dimensions, anatomical variations and clinical implications. METHODS: A systematic computer search of EMBASE, PubMed and Cochrane for literature related to the psoas valley was undertaken using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Clinical outcome studies, prospective/retrospective case series, case reports and review articles that described the psoas valley and its synonyms were included. Studies on animals as well as book chapters were excluded. RESULTS: Of the 313 articles, the filtered literature search identified 14 papers describing the psoas valley and its synonyms such as iliopsoas notch, a notch between anterior inferior iliac spine and the iliopubic eminence, Psoas-U and anterior wall depression. Most of these were cross-sectional studies that mainly analyzed normal skeletal hips. In terms of anatomical variation, 4 different configurations of the anterior acetabular rim have been identified and it was found that the curved type was the most frequent while the straight type may be nonexistent. Additionally, the psoas valley tended to be deeper in males as compared with females. Several papers established the psoas valley, or Psoas-U in a consistent location at approximately 3 o'clock on the acetabular rim which may have implications with labral pathology. CONCLUSION: This review highlights the importance of the anatomy of the psoas valley which is a consistent bony landmark. The anatomy and the anatomical variations of the psoas valley need to be well-appreciated by surgeons involved in the management of young adults with hip pathology and also joint replacement surgeons to ensure appropriate seating of the acetabular component.

Sense of coherence and self reported health amongst medical students: A cross sectional study.

BACKGROUND: In late 70s, Antonovsky proposed a salutogenic theory. This theory was based on the assumption that the human environment causes stress, and sense of coherence could serve as a stress-resisting resource. This study examined association between sense of coherence and self rated health of medical students. The aim of this study was to determine the association between sense of coherence and self reported health among medical students. METHODS: This was a cross-sectional analytical study conducted among medical students. The study tool used was a pre-designed, pre-tested, structured and self administered questionnaire (SOC scale and the SF 12 version 2 self-rated health measure). 172 medical students participated in the study. The data collected was entered into Microsoft Excel and analyzed using SPSS 20. All students were educated about sense of coherence after the study. RESULTS: Among the respondents, there were 99 (57.6%) female students, and 73 (42.4%) were male. The mean age of the students was 20.8 + 1.20 years. The mean (±SD) SOC scale score was 56.15 (±7.83). The mean self rated health score was 53.52 (±7.11). Pearson's χ2 test was used to determine the association and there was significant association between students SOC and self reported health (p < 0.000). CONCLUSION: There is a significant association between score of sense of coherence scale and self rated health among medical students.

Hindi and English Letter Cancellation Tasks : Does language have an effect on performance?

Letter Cancellation Tasks (LCTs) are paper-pencil based psychomotor tasks that are commonly used to assess cognitive functions of healthy volunteers and patients. The performance on LCTs can be affected by the proficiency in the language used for the tasks. This is of importance in a country like India where most of the population is not fluent in English. Since most of the cognitive tests are in English, the population that can be recruited for cognitive studies gets limited. It is therefore essential to develop tests in Indian languages like Hindi and compare the results obtained with tests in English. The present study evaluated the effect of Hindi and English language on performance of one, two and three letter cancellation tasks. The study was conducted on 50 healthy volunteers after taking written, informed consent. Subjects were asked to cancel out letters in One, Two and Three LCTs in Hindi and English language. The total time taken to complete each test and number of errors were statistically analyzed by unpaired t-test. The results revealed that a significantly longer time was taken to complete Hindi LCTs than English LCTs. The error rates in the Hindi LCTs were more than that in English with the difference in One Letter Cancellation Task being significant. This is probably because of the complex script for Hindi requiring more time and neural resources for processing.

Targeted SLNs for management of HIV-1 associated dementia.

CONTEXT: HIV-1 associated dementia (HAD) is an evolving disease in the category of neurological disorders. OBJECTIVE: Nifedipine-loaded solid lipid nanoparticles (SLNs) were developed and coated with Tween 80 to facilitate enhanced brain drug delivery for the treatment of HAD. MATERIALS AND METHODS: SLNs were prepared using solvent injection method. Lipids consisted of tristearin, hydrogenated soya phosphatidylcholine (HSPC) (1.5:1 w/w). Nifedipine was model drug in this study. Tween 80 (0.5% v/v) was taken as key modulator. SLNs were characterized for particle shape, size, zeta potential, entrapment efficiency, in vitro drug release, DNA fragmentation, cytotoxicity potential and in vivo studies. RESULTS: The SLNs (plain and coated) were found to be in nanometric in size (∼120 nm) with more than 70% entrapment efficiency. In vitro drug release profile reflected sustained release up to 48 h. Tween 80-coated SLNs showed higher percentage of DNA fragmentation in vitro and enhanced cell viability in sulforhodamine assay (rat cortical cells) as compared to plain drug and uncoated SLNs due to facilitated uptake of SLNs and reversal of P-gp efflux by virtue of Tween 80. Biodistribution study performed on vital organs, i.e. brain, heart, liver, spleen, lungs and kidney showed increased accumulation of Tween 80-coated SLNs in the brain. DISCUSSION AND CONCLUSION: Tween 80 enhanced localization of SLNs in the brain as compared to uncoated SLNs. This approach can be employed effectively to transport chemotherapeutics across the BBB for management of HIV-1 associated dementia and other ailments.

SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly.

FtsZ has been recognized as a promising antimicrobial drug target because of its vital role in bacterial cell division. In this work, we found that a taxane SB-RA-2001 inhibited the proliferation of Bacillus subtilis 168 and Mycobacterium smegmatis cells with minimal inhibitory concentrations of 38 and 60 µM, respectively. Cell lengths of these microorganisms increased remarkably in the presence of SB-RA-2001, indicating that it inhibits bacterial cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in B. subtilis 168 cells and also affected the localization of the late cell division protein, DivIVA, at the midcell position. Flow cytometric analysis of the SB-RA-2001-treated cells indicated that the compound did not affect the duplication of DNA in B. subtilis 168 cells. Further, SB-RA-2001 treatment did not affect the localization of the chromosomal partitioning protein, Spo0J, along the two ends of the nucleoids and also had no discernible effect on the nucleoid segregation in B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly and bundling of FtsZ protofilaments, and reduced the GTPase activity of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting that it does not bind to the GTP binding site on FtsZ. A computational analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region between the C-terminal domain and helix H7, and the binding site of SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001 inhibits bacterial proliferation by targeting the assembly dynamics of FtsZ, and this can be exploited further to develop potent FtsZ-targeted antimicrobials.

Study of paraCEST response on six-coordinated Co(II) and Ni(II) complexes of a pyridine-tetraamide-based ligand.

This study highlights the successful synthesis of a potential ligand, 2,2',2'',2'''-((pyridine-2,6-diylbis(methylene))bis(azanetriyl))tetraacetamide (PATA), along with its corresponding Co(II) and Ni(II) complexes for paraCEST-based agents. X-ray diffraction data confirmed that both the complexes are six coordinated with distorted octahedral geometries, but only the [Co(PATA)]2+ complex has a good structural feature to show paraCEST activity. After a thorough characterization of the ligand and both of its complexes, various studies, including solution-state magnetic properties, redox properties, temperature, and pH variation studies, were carried out. [Co(PATA)]2+ remained inert in the presence of competing ions, under acidic conditions, at high temperatures, and in the physiological pH range. The paraCEST response of [Co(PATA)]2+ has been measured in the presence of HEPES buffer medium, and a high paraCEST feature was discovered at both 37 and 25 °C. The pH variation paraCEST studies were carried out and the exchange rate constant of the probe at 37 and 25 °C was also determined. However, due to the fast exchange of water protons, the [Ni(PATA)(OH2)]2+ complex remained inactive in the CEST process.

Nuances in Growing Rod Surgery: Our Initial Experience and Literature Review.

Introduction: Growing rod construct is one of the most widely acknowledged treatment modalities for early-onset scoliosis around the world, but it is not without complications. Throughout the course of treatment, numerous planned and inadvertent surgical interventions are required, which increase the complexity of the treatment. We share our experience with case examples along with extensive literature search and review to get an insight and document the complications with growing rod treatment. Case Report: These cases underwent surgery with dual growing rod for thoracolumbar idiopathic scoliosis in the view of failed conservative treatment and progressive deformity. Superficial infection is in one case and recurrence of deformity was a common finding though correction of deformity and final fusion was achieved in the cases. Breakage of screws, autofusion of the spanned segments, and profuse bony growths over the implants are common finding to get. Fibrosis and scar tissue from the previous surgeries result in difficulty in the exposure and performing corrective osteotomy. Conclusion: Growing rod surgery has high complication rates. Repeated surgical and anesthesia exposure pose a great risk to the body and immature skeleton of the young patient. Previous studies have put forth many possible course of action to lower down the complication rates but have met with variable results. A better implant design and surgical efficacy are needed to cut down the number of complications and surgical interventions in growing rod surgeries.

Pyrophosphate sensing by a fluorescent Zn2+ bound triazole linked imino-thiophenyl conjugate of calix[4]arene in HEPES buffer medium: spectroscopy, microscopy, and cellular studies.

An in situ prepared Zn(2+) complex of triazole linked imino-thiophenyl conjugate of calix[4]arene, [ZnL], was demonstrated to be highly fluorescent in HEPES buffer solution. [ZnL] has been used as a chemo-sensing ensemble for the recognition of phosphates in general and pyrophosphates in particular among the eighteen different anions studied. The chemo-sensing behavior of the [ZnL] has been demonstrated through fluorescence, absorption, visual fluorescent color changes, ESI MS, and (1)H NMR titrations. Variations in the microstructural features of L, its zinc complex and the complex upon addition of PPi have been demonstrated through atomic force microscopy and transmission electron microscopy. Such studies have been extended to see the permeability of the conjugate into the HeLa cells by fluorescence microscopy. In accession, a reversible "write-read-erase-read" logic gate property of L has been demonstrated through a feedback loop in the presence of Zn(2+) and PPi.

Cd2+ complex of a triazole-based calix[4]arene conjugate as a selective fluorescent chemosensor for Cys.

An N,N-Dimethylamine ethylimino-appended triazole-linked calix[4]arene conjugate, L, has been synthesized and characterized, and its Cd(2+) complex has been isolated and characterized. The structure of [CdL] was established by computational calculation using B3LYP/LANL2DZ. Time-dependent density functional theory calculations were performed to demonstrate the electronic properties of [CdL]. This highly fluorescing [CdL] has been used to recognize Cys selectively among the 20 naturally occurring amino acids. [CdL] exhibits a minimum detection limit of 58 ppb for Cys, with reusability and reversibility being imparted to the system during sensing. Thus, the sensing of Cys was well demonstrated using various techniques, viz., fluorescence, absorption, visual color change, electrospray ionization MS, (1)H NMR, and live cell imaging experiments.

A Zn2+ specific triazole based calix[4]arene conjugate (L) as a fluorescence sensor for histidine and cysteine in HEPES buffer milieu.

A highly fluorescent Zn(2+) complex of the triazole linked salicyl-imino-thiophenyl conjugate of calix[4]arene, [ZnL] has been demonstrated to be a chemo-sensing ensemble for the recognition of His and Cys among the naturally occurring amino acids in HEPES buffer milieu. The recognition behaviour of the [ZnL] towards these amino acids has been shown on the basis of fluorescence, absorption and visual fluorescent colour changes. The species of recognition were shown by ESI MS titrations, AFM & TEM microscopy and cell studies.

Imino-phenolic-pyridyl conjugates of calix[4]arene (L1 and L2) as primary fluorescence switch-on sensors for Zn2+ in solution and in HeLa cells and the recognition of pyrophosphate and ATP by [ZnL2].

Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L(1) and L(2), were synthesized and characterized. These two conjugates were shown to be selective and sensitive for Zn(2+) among the 12 metal ions studied in HEPES buffer medium by fluorescence, absorption, and visual color change with the detection limit of ~31 and ~112 ppb, respectively, by L(1) and L(2). Moreover, the utility of the conjugates L(1) and L(2) in showing the zinc recognition in live cells has also been demonstrated using HeLa cells as monitored by fluorescence imaging. The zinc complexes of L(1) and L(2) were isolated, and the structure of [ZnL(1)] has been established by single-crystal XRD and that of [ZnL(2)] by DFT calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc complexes. The isolated zinc complexes, viz. [ZnL(1)] and [ZnL(2)], have been used as molecular tools for the recognition of anions on the basis of their binding affinities toward Zn(2+). [ZnL(2)] was found to be sensitive and selective toward phosphate-bearing ions and molecules and in particular to pyrophosphate (PPi) and ATP among the other 18 anions studied; however, [ZnL(1)] was not sensitive toward any of the anions studied. The selectivity has been shown on the basis of the changes observed in the emission and absorption spectral studies through the removal of Zn(2+) from [ZnL(2)] by PPi. Thus, [ZnL(2)] has been shown to detect PPi up to 278 ± 10 ppb at pH 7.4 in aqueous methanolic (1/2 v/v) HEPES buffer.