FI behaviors were significantly different between countries, with the USA more likely to consume fluids to meet a total target volume  and consume fluid at the same time as structured daily activities  compared to FI alongside food intake. However, there were no differences in the types of beverage consumed , total fluid volume, and physical activity (PA) between countries (p > 0.05). Beverage consumption was higher among USA than AUS residents for water, beer, and wine (p < 0.05). Total fluid consumption was greater among males  than females , individuals who regularly consumed fluid during the day to meet a targeted volume , and those who regularly consumed fluid as a habitual behavior  compared to those individuals who only consumed fluid alongside mealtimes . FI behaviors differed between the USA and AUS; however, total volume consumed, type of beverage consumed, and FI beliefs were similar. FI practices and behaviors appear to be individualized and context-specific among the studied populations.Minimal data exist exploring intercontinental differences in fluid intake (FI) beliefs and behaviors and the impact on fluid intake practices . Therefore, this study explored the impact that FI beliefs and behaviors had on FI practices among emerging adults living in the United States (USA) and Australia (AUS). A total of 489 individuals . Participants detailed their FI practices. FI beliefs were evaluated to determine their contribution to FI behaviors across the day. Multinomial and multiple linear regression analyses explored the association of daily FI beliefs and behaviors across multiple domains. Independent sample  Compelling evidence suggests that meeting daily FI recommendations is associated with positive outcomes related to renal, cardiovascular, and metabolic health  occurs Due to the differences in individual physiological requirements, values referencing adequate intake (AI) are used as the dynamic and complex nature of body water regulation, and homeostasis prohibits the determination of exact hydration levels. Fluid intake recommendations have been disseminated by many leading public health organizations  indicate that sugar-sweetened beverage consumption is decreasing while water intake is increasing  driving FI behaviors in various environments. Textural elements of fluid  may contribute to the consumption of a particular beverage type and guide preferences for FI behaviors  were recruited to complete an online survey  to capture their FI beliefs and behaviors toward FI practices. University email listservs (University in the Southeastern USA), social media posts, and convenience sampling were used to recruit participants from November 2020 to June 2021. The study was intended to capture the practices employed by a diverse group of individuals with varying levels of daily energy expenditure, environmental conditions, health status, and occupations. Consent of the participants to complete this survey was implied by them clicking on the “I give consent to partake in the research study” button located at the bottom of the study information page before being able to view and complete the survey. All participants gave informed consent before undertaking the survey. This study was approved by the institutional review board at the University of North Carolina at Greensboro (#21-0132) and Queensland University of Technology (2000000945).To the authors' knowledge, there was no previous validated survey instrument to explore the research question. The lead investigator (JS) created an original questionnaire constructed from a variety of recent publications  participant characteristics and demographics, (2) FI practices, and (3) FI beliefs and behaviors.The first section of the survey sought to capture a greater understanding of the studied population. Participants were asked to select their year of birth (age) and their sex. Participants were also asked to detail their current country of residence and postcode/ZIP code of that residence and the country where they spent >50% of their early years of life (up to the age of 18 years). These items were added to explore heritage and environmental changes related to geography. Physical activity was captured by participants detailing their frequency  and duration  for light, moderate, and vigorous activities, and participants were provided exemplar activities which was adapted from the World Health Organisation . PhysicaThe second section of the survey was designed to explore the current practices implemented in everyday living. Participants were provided with images, and information detailed the volume (in ml and ounces) of one serving of the following beverages: water, juice, sugar-sweetened beverages , coffee, tea, wine, and beer. Participants were asked to indicate the amount of serves of each beverage consumed on average each day. Data were extrapolated to determine total fluid consumed for each beverage and total fluid intake over the day. Participants were asked to confirm consumption of beverage types (with extended examples) and included an option to indicate consumption of beverages not consumed.Fluid frequency was assessed by participant indicating the most appropriate response: “I consume fluid predominately at meal times only,” “I consume fluid regularly during the day when I feel I need it,” “I consume fluid regularly throughout the day as I have a total target consumption that I aim to meet,” “I predominately only consume fluid when I have structured activities throughout my day ,” “I consume fluid regularly throughout my day as it is a habit,” and “I don't know when I typically fluids.”The role of fluid imbalance and risk of acute and chronic health was assessed by asking the participants to indicate which physiological systems  would have increased risk if fluid imbalances were to occur.FI behaviors and how they relate to physiological mechanisms were assessed using 5-point Likert scale (strongly-agree to strongly-disagree). The five items included: (1) individuals with chronic health conditions typically require alterations to their previous practices of fluid intake volumes and frequency, (2) I regularly wake up during the night and require the bathroom for urination, (3) I notice a difference in taste between types of water , (4) Do you believe that lack of fluid intake impairs your cognitive function ?, (5) My regular fluid intake regime has decreased as I have aged.The final seven questions were structured as per the Precaution Adoption Process Model, and participants indicated their current beliefs toward the behavior from seven possible responses. Participants were asked their (1) belief about fluid volume consumption in relation to their physiological needs  as well as a subsequent question on (2) how the types of fluid may also impact those physiological systems, (3) the interaction between fluid imbalances and acute health conditions, such as cognitive alertness, tiredness, alterations in mood, increased body temperature, and headaches, (4) their beliefs toward fluid imbalances leading to chronic health conditions, such as chronic kidney disease, high blood pressure, and respiratory disorders, (5) how the types of fluid may also contribute to chronic health conditions, (6) their beliefs about how fluid intake can impair sleep (7), and finally, how fluid intake behaviors may change with aging. All items related to fluid intake and physiology and fluid intake behaviors were displayed in a randomized order to minimize the effect of health model ordering influencing responses.n = 3) with expertise in the fields of hydration and human performance. The instrument was trialed among a group of 10 emerging adults with various education levels to ensure adequate readability. The final instrument comprised of 23 items using Likert scale and multiple-choice response options. Due to the novel nature of the instrument, a true power analysis was unable to be determined. However, similar hydration survey instruments have shown adequate response rates when the total sample size is >300 responses and/or five times the number of instrument items   analyses were used to assess the differences in fluid intake consumption , physical activity, and outcomes of meeting FI recommendations between countries and sex differences within countries. Multinominal and multiple linear regression analyses were used to assess associations of fluid intake and associated beliefs and behaviors. All regression coefficients were presented relative to changes in fluid volume with their associated confidence intervals. Alpha was set at p < 0.05 for all analyses.Analyses were performed in R  using the RStudio Environment (Version 1.0.143) and SPSS . All data were presented as mean and standard deviation (SD) unless otherwise specified. The n = 6, 1.1%) from the analysis due to the lack of adequate sample size. The remaining 15 (2.7%) participants were removed due to the reasons associated with location of residency, falsified responses , or age <18 years (n = 3) who reported fluid intake volumes >7 L/day were only excluded from the analysis of fluid consumption evaluations as their reported values are two times greater than recommended values and likely an error in self-reporting. Participant demographics are detailed in The survey was completed by 548 individuals with a total of 489 individuals included in the final sample. A total of forty-seven (8.6%) individuals were removed from the analysis due to non-completion of survey >67%. The cutoff score (>67%) was chosen as the final seven items of the survey were developed to reflect a health behavior model, and to ensure that adequate response to most survey items was completed. Individuals who did not identify as male or female sex were removed (18 years . Particin = 54) of males and 48% (n = 143) of females in the USA, respectively. Similarly, 54% (n = 18) of males and 48% (n = 33) of females in the AUS sample population met the AUS fluid intake guidelines (t(295), 3.324; p ≤ 0.001 and male 3,319.30 ± 2,717 ml; t(91), 2.891; p = 0.002]. Furthermore, total FI in this USA sample population was statistically greater than the current USA FI guidelines . Total FI was greater among all males  than females . Plain water intake contributed to an average total of 1,585 ± 1,248 ml across the participants and was significantly different between locations with greater consumption in USA participants .There were no differences in the type of beverage consumed , total fluid volume, PA, or FI beliefs between countries (t(487), 2.660; p = 0.008] and water intake  were significantly higher with those who completed >150 min of MVPA per week.Fluid volume  and water intake  greater in those who completed >150 min of moderate–vigorous physical activities per week. Observational trends in those who meet the Physical Activity Guidelines for Americans show a 9.6% increase from 1998 to the latest report released by the CDC in 2018  should be considered as this could assist in improving overall health outcomes which is the ultimate basis of FI recommendations.The data is available upon reasonable request.The studies involving human participants were reviewed and approved by University of North Carolina at Greensboro and Queensland University of Technology. The patients/participants provided their written informed consent to participate in this study.JS, JH, and WMA contributed to study design, data collection, and interpretated the data. JS and TA contributed to data analysis and data visualization. All authors contributed to critical revisions of the manuscript and have read and agreed to the published version of the manuscript.This work was the authors' own and not that of the United States Olympic and Paralympic Committee, or any of its affiliates or members.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acinetobacter baumannii-calcoaceticus species complex (ACB) and Stenotrophomonas maltophilia (SM) are opportunistic, non-fermentative organisms that can cause serious hospital-acquired infections in immunocompromised patients. These pathogens are inherently resistant to several common drug classes and often acquire other resistance mechanisms, making them difficult to treat. In this study, we analyzed the susceptibility of contemporary ACB and SM isolates to minocycline (MIN), levofloxacin (LEV), meropenem (MER) for ACB, and trimethoprim-sulfamethoxazole (T/S) for SM. Isolates were collected as a part of the SENTRY Antimicrobial Surveillance Program from 2014-2021.Susceptibilities of SM and ACB to MIN and comparators, 2014-2021Isolates were collected from hospitalized patients in 35 US medical centers. Hospitals submitted 1 isolate per patient per infection episode that met local criteria for being the likely causative pathogen. Identification was performed by the submitting laboratory and confirmed by JMI Laboratories with matrix-assisted laser desorption ionization-time of flight mass spectrometry or other standard methods as required. Isolates were tested for susceptibility (S) to MIN and comparators using the CLSI broth microdilution method. All infection types were included in the analysis. CLSI (2022) breakpoints were applied.A total of 1,029 ACB and 1,520 SM were tested. Pneumonia in hospitalized patients was the most common infection from which ACB (57.0%) and SM (73.9%) were isolated. The %S of the agents tested against the organisms in this study are shown in the table. MIN had the highest %S for ACB (86.2%S) and SM (99.5%S). The %S of ACB and SM to all 3 agents varied over the period studied. MIN %S to ACB decreased in 2020 (80.6%) but rebounded in 2021 (86.2%). LEV and MER showed an overall trend of increasing S for ACB, with slightly lower %S in 2020-2021. SM had stable %S to MIN and T/S . LEV varied from 84.3%S (2015) to 69.2%S (2018).in vitro data suggest that MIN is a useful treatment option for infections caused by ACB or SM.%S to MIN remained stable and higher than other agents tested for both ACB and SM, pathogens which have limited therapeutic alternatives. ACB showed < 6% decrease in %S to all 3 agents in 2020-2021. These Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.


Motivated by the growing demand for target chemosensors designed with diagnostic or therapeutic capability for fibrils related to amyloidosis diseases, we investigated in the present work the response mechanism of dicyanomethylene-based fluorescent probes for amyloid fibril using a combined approach, including molecular docking, quantum mechanics/molecular mechanics (QM/MM), and the quantum chemical method. Various binding modes for the probes in β-amyloid (Aβ) are discussed, and the fibril environment-induced molecular optical changes at the most stable site are compared to the fibril-free situation in aqueous environments. The results reveal that the fluorescence enhancement for the probes in Aβ observed experimentally is an average consequence over multiple binding sites. In particular, the conformational difference, including conjugation length and donor effect, significantly contributes to the optical property of the studied probes both in water and fibril. To further estimate the transition nature of the molecular photoabsorption and photoemission processes, the hole-electron distribution and the structural variation on the first excited state of the probes are investigated in detail. On the basis of the calculations, structure–property relationships for the studied chemosensors are established. Our computational approach with the ability to elucidate the available experimental results can be used for designing novel molecular probes with applications to Aβ imaging and the early diagnosis of Alzheimer’s disease. Dementia is now the seventh leading cause of mortality globally, and it has one of the highest costs to society . AlzheimN,N-dimethylaminophenyl-donating group and dicyanomethylene-accepting group bridged by π-conjugated double bonds. It showed that the probe with the longest polyenic chain exhibited high affinity and maximum emission in Aβ plaques [A variety of detection methods have been reported for the analysis of active substance amyloid related to AD ,8,9,10.  plaques . In orde plaques . Moreove plaques . In addi plaques . Kim des plaques .Although the widespread experimental measurements emphasize the high affinities and excellent fluorescent properties for the dicyanomethylene-containing Aβ probes, it is necessary to have an in-depth understanding of the nature of interactions between the biostructure and sensors, further revealing the inner mechanisms. More importantly, there is increasing demand for comparing the properties of the probes at the same level. Based on this knowledge, one can propose the structure–property relationship and further provide design strategies for Aβ fluorescent probe with highly sensitive and rapid fluorescence response. Motivated by these scientific issues, theoretical investigations are highly desired. However, the research on this aspect is deficient on account of the computational limitation of simulating such a multiscale fibril–ligand complex. In this work, an integrated approach, including molecular docking, quantum mechanics/molecular mechanics (QM/MM) method, and quantum chemical calculation, is employed to study the interaction of the fibril–probe complexes and amyloid-specific optical properties of versatile dicyanomethylene-based fluorescent probes ,23,24,25The primary goal of this work is to study the interaction of dicyanomethylene-based fluorescent probes with Aβ fibril and to investigate the β-amyloid specific optical properties of the probes to elucidate the responsive mechanism. Fourteen substituent groups were chosen as the electron donor to connect with the dicyanomethylene moiety through the polyene chain. By varying the length for the polyene spacer, a total of 32 probe models which had been synthesized and characterized experimentally ,23,24,25Docking simulations show that the studied chemosensors can be mainly docked onto six different sites in the fibril shown in . Thereind) has been reported to be 1.95 μM, indicating that Site-1 is probably responsible for the probe binding in nature. Thus, one can judge that Site-1 is the dominant binding site, while other binding sites play incidental roles. For each series of the investigated probe in the same site, the binding energy is much lower for the probe with a longer polyene chain than that with a shorter chain. For example, binding energies for probes 1 to 5 in Site-1 are −7.88, −9.08, −9.88, −11.01, and −11.73 kcal/mol, respectively; and, for probes 6–8, the values are −7.53, −8.66, and −9.46 kcal/mol, respectively. This is in good agreement with experimental observations that probes with longer conjugated π system display a higher affinity to Aβ [Clustering of multiple binding sites with binding energy for each binding mode is collected in ty to Aβ . TherefoBy counting the binding sites for all probes in Aβ see B,C, the fω and αμ denote the excited energy of the state f and the electric dipole moment operator in the direction α, respectively. To elucidate the character of the photoabsorption of the probes both in aqueous and in Aβ, we carried out optical property calculations using two different approaches, namely, the quantum chemical method and the QM/MM framework, as mentioned above.When studying the photophysics processes, including absorption and emission, oscillator strength defined as 2O, and 495 and 464 nm in Aβ, respectively, as plotted in 2O and in Aβ. However, the variation tendency of the absorption intensity is inconsistent with the increase in the spacer length.In all cases except for probe 31, the absorption spectra in both aqueous and fibril exhibit one intense band in the UV or visible region, while probe 31 possesses two absorption bands locating at 550 and 509 nm in H2O. Upon binding with Aβ, the absorption maxima are blue shifted and the intensity is reduced in comparison with those in aqueous on account of the interaction between the molecule and the fibril. The calculated wavelength corresponding to the vertical electronic transition for selected probes is compared to the experimental values on the absorption maxima in δ, the absorption intensity is determined by the transition dipole moment and the excitation energy. The data on 0fμ and δabs listed in To further reveal the character of electron transition involved in the absorption, the electronic dipole moment and the transition natures of the probes are collected in By utilizing the useful tool of hole–electron analysis, the electron excitation process for the probes is graphically shown in Concerning the application as fluorescent probes, it is desirable to have an intense fluorescent response for the probe when binding with Aβ. For this reason, the excited state dynamics are investigated by optimizing the first excited state for the probes in aqueous and in Aβ fibril. Upon excitation, the molecule will absorb the photon energy and one electron will be transferred, leading to differences in molecular geometry and electronic structure. Conformational changes between different states for partial probes are quantitatively described by RMSD as shown in 2O are also given in Since the conformation is extraordinarily sensitive toward the molecular optical properties, the photoemission properties of the studied probes in Aβ are subsequently explored and the results in H2O, respectively, and the values shift to 548, 512, 483, and 542 nm in the fibril. This indicates the fibril environment induced changes and a donor effect on the emission properties of the probes, following the case of our previously reported results [2O corresponds to 522, 709, and 700 nm, respectively, and blue shift to 491, 648, and 594 nm in Aβ. It is notable that the maximum emission wavelength for probe with a similar architecture but an extended polyene chain exhibits a bathochromic shift with enhanced absorption intensity in H2O. Due to the interaction with the fibril, the fluorescent peak shows a blue shift and the intensity is reduced compared with those in aqueous. Overall, the conformational and the microenvironment significantly contribute to the fluorescent property of the probes, which is consistent with the experimentally reported observations on the trend [2O are convergent, while probe 16 shows an evident discrepancy both in H2O and Aβ. The difference in convergence for different probes probably originates from the PCM model only covering a long-range interaction, while ignoring the vibrational contribution, the hydrogen bond with the solvent, and the complicated interaction between molecules and all binding sites, etc. Especially, the fluorescence enhancement was observed in the corresponding experimental works, whereas the calculated photoemission intensity in Aβ was smaller than that in water. Thus, restricted geometry changes in the fibril microenvironment can be determined and the favorable planarity of the probes is achieved in an aqueous solution. It also should be noted that only the most likely binding site with the largest binding affinity is taken into consideration in the calculation. In fact, various binding sites for a probe are available, and the average intensity over all sites may be experimentally measured.The emission peak calculated for probes 3, 8, 11, and 13 are 629, 611, 581, and 636 nm in H results . The sithe trend ,23,24,25τ = c3/(2Eemi2δemi) are listed in Eemi and δemi denote the transition energy and oscillator strength, respectively. A large Stokes shift in the Aβ probes are demonstrated both in H2O and in the fibril, thus the overlap between absorption and emission spectra is small, avoiding the reduction of fluorescence efficiency caused by energy transfer. In addition, the emission lifetime for the probe in the fibril is located in the nanosecond time domain. Thus, a fast emission process can be expected, which results in facility of fluorescent detection for Aβ. Although the fluorescence peak is blue shifted in the fibril for the probes except for probe 29, the fluorescent oscillator strengths are decreased, while that of probe 29 is increased  ased see . TherefoFor the purpose of illustrating the transition natures in the emission process, the hole and the electron distributions for probes 3, 20, and 29 are calculated representatively based on the first excited state geometry. The corresponding heat maps are presented in On the basis of the dynamical analysis on the excited state, we tried to establish the structure–property relationship for the probes. Firstly, extension of π-conjugation can largely affect the fluorescence wavelength. In general, a red shift is observed in emission maximum with the increase of spacer, which has been experimentally reported elsewhere . For exa2O is used in the calculations. Considering the information of binding sites was not available, we carried out blind molecular docking for the probes with full-length β-amyloid (PDB code: 5OQV) [Geometry optimization and electronic structure calculation for the molecular probes were performed using the density functional theory (DFT) method with the B3LYP functional and 6–31+G basis set using Gaussian 16 . Frequene: 5OQV)  using AUe: 5OQV) . The numFor complexes with the highest binding affinity among all configurations, the QM/MM method with a two-layer ONIOM model was adopted to optimize the geometry and to calculate the optical properties . In thisix, iy, iz and i in different conformations and N is the total number of atoms in the molecule. For the first excited state, the heat maps of the hole and the electron distributions are plotted. All of these analyses were realized by Multiwfn [In order to illustrate the electron excitation characteristics, geometry changes between different states of the molecule as well as the hole-electron distributions are analyzed based on the optimized structures of the probes. Thereinto, the root mean squared displacement (RMSD) with the expression Multiwfn ,33.λ) in different microenvironment for the studied probes is 61~106 nm, indicating the contribution of donor moiety on molecular photophysical properties. These results are in good agreement with the experimental measurements. To better understand the transition mechanism, the hole and the electron distributions are calculated based on the excitation process, which shows the variation on charge transfer for the probes with different electron-donating groups or π-conjugate length. Based on the results, an inner structure–property relationship for the studied probes is revealed. It is proposed that the comprehensive consideration on binding affinity, extension of π-conjugate, and the electron donating/accepting ability of the fluorophore should be combined to improve the efficiency of the dicyanomethylene-containing Aβ fluorescent probe. Importantly, the calculations indicate that naphthalene derivative can act as a preferable electron-donating group in the studied probes. Our work validates the feasibility of the employed integrated approach on capturing the fibril-induced changes in structure and optical properties of Aβ molecular probes, and the findings can bring a necessary theoretical insight on designing novel molecular probes for detecting amyloid fibrils in Alzheimer’s disease.In this work, an integrated approach including molecular docking simulation, quantum chemical calculation and QM/MM method is adopted to systematically study the fibril-induced optical properties of versatile dicyanomethylene-based fluorescent probes for β-amyloid in Alzheimer’s disease. The important binding sites for the probes in the Aβ fibril are identified and binding modes are characterized to make a comprehension on the interaction between the fibril and the molecule. The conformation of the ligand–fibril complexes with a higher binding affinity are selected for optical property investigations. On the basis of the QM/MM calculations, lengthening the spacer tends to significantly induce a red shift in the optical spectra, which reveals the importance of molecular size on the optical property of the probes. In addition, the absorption maximum for probes with the same polyene chain ranges from 595 nm to 478 nm, corresponding to the probe with naphthalene ring and with a chalcone scaffold as the electron donor, respectively. Similarly, the shifts of the fluorescent wavelength (Δ


We provide an updated review of clinical trials evaluating the combination of BRAF/MEK inhibitors with anti-PD-(L)1 therapy (triplet therapy) for patients with advanced BRAF-mutant melanoma, accompanied by a summary of the biological evidence supporting this combination.Resistance to BRAF/MEK inhibition and comparatively low response rates to immune checkpoint inhibitors remain clinical challenges in the treatment of melanoma. Preclinical data demonstrates that targeted therapy is immune-modulatory and synergises with immune checkpoint inhibition. Several randomised controlled trials have evaluated the combination of targeted therapy with immune checkpoint inhibition.Triplet therapy has shown improvements in progression-free survival and durability of response compared to BRAF/MEK inhibition alone; however, questions remain regarding the best clinical scenario for implementation of this regimen in the era of front-line immunotherapy. Current strategies for the treatment of advanced BRAF-mutant melanoma include targeted therapies in the form of BRAF/MEK inhibitors (BRAF/MEKi) and immune checkpoint inhibitors (CPI), including antibodies to programmed cell death-1 (anti-PD-1), programmed cell death ligand-1 (anti-PD-L1) and cytotoxic T lymphocyte antigen (anti-CTLA4). Targeted therapies are characterised by the induction of rapid but often short-lived responses whilst CPI have led to unprecedented long-term disease control, albeit with lower response rates. Preclinical models and translational data demonstrate that BRAF/MEKi have an immunomodulatory effect on the tumour microenvironment (TME), further strengthening the biological rationale of combining the complimentary clinical profiles of BRAF/MEKi and CPI. In recent years, the combination of BRAF/MEKi and CPI has been evaluated in several randomised controlled trials (RCTs). Here, we provide an updated review of available clinical data and an accompanying summary of the biological basis of combined BRAF/MEKi and CPI, so-called triplet therapy.BRAFV600 mutations occur in approximately 35–50% of cutaneous melanoma and result in constitutive activation of the MAPK pathway . BRAF-muIn addition to their direct effects on tumour cells through blockade of the MAPK pathway, BRAF/MEKi have also been shown to modulate the TME . PreclinIn contrast, an immunosuppressive TME at the time of progression on BRAF/MEKi therapy has been observed in both preclinical and clinical studies. Resistance to BRAF/MEKi is associated with upregulation of PD-L1 on both melanoma cells and immune cells , 22, a r(−/−) melanoma mouse model demonstrating prolonged survival and slowed tumour growth in mice treated with combined BRAFi and anti-PD-1 or anti-PD-L1, whereas anti-PD-(L)1 monotherapy failed to induce response [To test the hypothesis of improved anti-tumour immunity with triplet therapy, preclinical studies have examined co-administration of BRAF/MEKi and anti-PD-1 or anti-PD-L1 antibodies in melanoma mouse models. In a syngeneic mouse model of SM1 BRAFV600E mutant melanoma, triplet therapy demonstrated heightened anti-tumour activity compared with either anti-PD-1 monotherapy or anti-PD-1 in combination with either BRAFi or MEKi alone , 27. Thiresponse . In bothresponse , 26. Higresponse , 26. In response . OverallObservations from early phase clinical trials have assisted in refining this biological hypothesis and have informed the design of current clinical trials. Translational studies have sought to characterise immune cell infiltrates of serial tumour biopsies collected from melanoma patients on treatment and at the time of progression. One such study of patients treated with a BRAF inhibitor showed an early increase in CD8 + T-cell infiltrates after 3–15 days on treatment, which correlated with a decrease in tumour size , demonstInitial phase I clinical trials of triplet therapy explored the safety and feasibility of combining BRAF/MEKi with the anti-CTLA4 antibody ipilimumab. This combination resulted in prohibitive gastrointestinal toxicity. Dose-limiting hepatotoxicity was observed in 6 of 10 patients treated with the combination of vemurafenib and ipilimumab  and coliThree RCTs have evaluated the combination of BRAF/MEKi with either anti-PD-1 or anti-PD-L1 antibodies in patients with unresectable or metastatic melanoma. A fourth RCT is ongoing  comparing encorafenib, binimetinib and pembrolizumab to pembrolizumab monotherapy is yet to be reported.Part 3 of the KEYNOTE-022 phase I/II clinical trial randomised 120 patients with unresectable stage III or IV BRAFV600E or V600K mutant melanoma to receive dabrafenib 150 mg BID and trametinib 2 mg daily in combination with either pembrolizumab 200mg 3-weekly or placebo . The phap-value of 0.0249. KEYNOTE-022 failed to reach significance at the pre-specified analysis but notably achieved a HR of 0.53 in the 36-month post hoc analysis, suggesting longer follow up is required to demonstrate the true benefit of triplet therapy compared to doublet therapy. The control arm in COMBI-i performed somewhat better than expected .Each of the three RCTs reported to date have yielded numerically similar results with respect to their uniform primary endpoint of investigator-assessed PFS with results of 16.9, 15.1 and 16.2 months in the triplet arms compared to 10.7, 10.6 and 12.0 months in the placebo arms of KEYNOTE-022, IMspire150 and COMBI-i, respectively. However, only the IMspire150 trial demonstrated a statistically significant improvement in PFS with a hazard ratio (HR) of 0.78 and a ORR, duration of response (DOR) and overall survival (OS) were key secondary endpoints across the three trials. Although the pembrolizumab arm of KEYNOTE-022 surprisingly showed a lower ORR (63%) compared to the control arm (72%), this finding was not replicated in either of the phase III trials, and may have been due to the higher proportion of stage M1c patients in the triplet arm of this study.Importantly, the duration of response was demonstrably longer with triplet therapy across all three studies Table , supportSubgroup analyses demonstrated a consistent trend towards improved PFS with triplet therapy across patient age, performance status, LDH level and disease stage across all three trials. The COMBI-i trial has reported a biomarker analysis evaluating PFS according to tumour mutational burden (TMB). Two hundred thirty-seven patients had low TMB (defined as < 10 mutations per megabase) and 177 patients had high TMB (10 or more mutations per megabase). A greater numerical improvement in PFS  was seen in the high TMB group but this did not meet statistical significance .Improvements in durability and efficacy are offset to an extent by an increase in toxicity observed in the combination arms of all three trials. Increased rates of fever, rash, diarrhoea and liver transaminase elevation were observed with triplet regimens. Substantially higher rates of treatment-related grades 3–5 toxicity were seen in the triplet arm compared to the doublet arm of KEYNOTE-022 and COMBI-i . There was one treatment-related death (due to pneumonitis) in KEYNOTE-022. Although the reported rates of treatment-related grades 3–4 toxicity in IMspire150 exceed 70%, toxicity profiles between the combination and placebo arms were comparable , and largely driven by asymptomatic and reversible laboratory abnormalities, including elevated creatine phosphokinase, and the rate of treatment discontinuation in this trial was low . Similarly, discontinuation for treatment-related adverse events in COMBI-i was low at 12.4%  compared to 8% (control arm). In contrast, toxicity of the triplet regimen in KEYNOTE-022 led to discontinuation in 47% of patients , compared to 20% in the control arm.Whilst only one of the three randomised controlled trials to date has met its primary endpoint in terms of a statistical improvement in PFS, the strikingly consistent numerical increase in PFS and duration of response in the triplet arms does support a role for the addition of anti-PD-1 to BRAF/MEK inhibitors. Where these triplet regimens will sit in the clinical armamentarium has been challenged by the evolution of combination ipilimumab and nivolumab (IPI + NIVO) as the current front-line standard of care for unresectable melanoma , 9, 38. There may be a role for triplet therapies in patients for whom IPI + NIVO is contraindicated or undesirable given the high rates of grades 3–4 immune-mediated toxicity associated with IPI + NIVO. Whilst the toxicity of triplet therapy is not insubstantial, the profile is arguably more tolerable than that of IPI + NIVO, with the exception of the KEYNOTE-022 regimen , 35, 36.These trials demonstrate that the combination of BRAF/MEKi/CPI is a feasible and active treatment option. In the context of an evolving treatment landscape with multiple immune checkpoint combinations now available, the future of the triplet regimen will depend on identifying the ideal clinical scenario for implementation, which may be in well-defined patient sub-populations or beyond the front-line metastatic setting.The TRIdent study is a single-arm phase II study of dabrafenib, trametinib and nivolumab that notably included anti-PD-1 refractory patients and patients with brain metastases who were BRAF-mutant and BRAF/MEKi-naïve. An objective response rate of 88% (14 of 16 patients) was achieved in the anti-PD-1 refractory group, and an intracranial response rate of 57% was seen in patients with brain metastases .Triplet therapies may have a future role in the adjuvant or neoadjuvant setting. Both BRAF/MEKi and anti-PD-1 have demonstrated efficacy in this area –43. The The observation from preclinical and translational data that the effect of BRAF and MEK inhibitors on the tumour microenvironment is dynamic and temporally dependent raises the possibility that sequential administration of BRAF/MEKi and CPI could optimise anti-tumour immunogenicity. As yet, there is no completed randomised, prospective data to inform the optimum sequence of BRAF/MEK inhibitors and CPI therapies in patients with BRAF-mutant melanoma. The recently presented interim results from the DREAMseq trial indicate improved 2-year PFS and OS rates with upfront IPI + NIVO followed by dabrafenib and trametinib on progression, compared to the reverse sequence . HoweverCombining the clinically diametric profiles of BRAF/MEK inhibitors and CPI is supported by strong biological rationale. Namely, an immune-stimulatory effect of BRAF/MEKi, implication of immune-exhaustion in acquired resistance to BRAF/MEKi and synergy between BRAF/MEKi and CPI in preclinical models have all been observed. Three randomised clinical trials evaluating combination BRAF/MEKi and CPI in the first-line setting demonstrate strikingly similar numerical and clinically significant improvements in progression-free survival and durability of response compared to targeted therapy alone. The clinical application of triplet therapies in the first-line metastatic setting has been limited by a lack of comparison to either single-agent anti-PD-1 or IPI + NIVO, which have since emerged as standards of care for both BRAF-mutant and wild-type melanoma. Nonetheless, both preclinical and clinical data to support the triplet regimen is compelling. Investigation of how best to apply this synergy clinically is ongoing and includes evaluation in the (neo)adjuvant setting as well as investigation of sequential and alternating therapies. Such research must include translational objectives and patient biospecimens to deepen our understanding of the interaction between BRAF/MEKi, CPI, tumour characteristics and the immune microenvironment to direct patient selection or to unveil novel therapeutic strategies.


The reservoir for zoonotic o’nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats. Togaviridae, Genus: Alphavirus) are closely related mosquito-transmitted viruses endemic to Uganda [Aedes africanus  mosquitoes collected in Zika forest near Entebbe [Both o’nyong-nyong virus (ONNV) and chikungunya virus (CHIKV)  and 010-015 . Bats were captured using harp traps or mist nets, taking appropriate biosafety precautions. Upon capture, bats were placed individually in holding bags. Blood from bats captured in Maramagambo forest was collected and stored as described by Towner et al. . Bats fr> 4-fold higher than the ONNV titer was considered CHIKV-positive. Any bat with a neutralizing antibody titer for ONNV that was higher than that for CHIKV was considered ONNV-positive, with a minimum ONNV-positive titer as PRNT80 = 10, as long as the corresponding CHIKV titer was < 10. Samples with neutralizing antibody titers for CHIKV that were < 4-fold higher than titers for ONNV, or those with equal titers for CHIKV and ONNV were considered non-specific alphavirus-positive. Mouse hyperimmune ascites fluid generated against either CHIKV or ONNV was obtained from the CDC Arbovirus Reference Collection for use as positive control antisera.All serum samples were frozen at −80 °C until they were heat inactivated and tested for neutralizing antibodies against CHIKV and ONNV by plaque reduction neutralization test (PRNT) . AlphaviRousettus aegyptiacus), 82 little free-tailed bats (Chaerephon pumilus), 15 African straw-colored fruit bats (Eidolon helvum), 99 Ethiopian epauletted fruit bats (Epomophorus labiatus), 9 Angolan rousette bats (Myonycteris angolensis), 33 Angolan mops bats (Mops condylurus), 10 Cape long-eared bats (Nycteris thebaica), and 4 Noack’s leaf-nosed bats (Hipposideros ruber)  . Forty os ruber) .Here, we report exposure of one cave population of Egyptian rousette bats in Uganda sampled during 2009 to both ONNV and CHIKV, and a single little free-tailed bat captured near Entebbe during 2011 to ONNV, determined by the detection of specific neutralizing antibodies in bat serum.Pteropus pliocephalus) was possible, although viremia in the bats was low [Artibeus lituratus) and two Seba’s short-tailed bats (Carollia perspicillata) in Trinidad by hemagglutination inhibition [Many reports exist of alphavirus detections, isolations, and seroprevalence in different bat species around the world ,31, demo was low , and anthibition . These eDuring 2009, approximately 13% of the Egyptian rousette bats in Python cave, Maramagambo forest, had neutralizing antibodies to ONNV, 0.7% had antibodies to CHIKV, and an additional 2.6% had neutralizing antibodies with non-specific alphavirus cross-reactivity based on our criteria. Even though ONNV has not caused an epizootic in Uganda since 1996–1997, the virus may be circulating at low levels during this interepidemic period. Kasokero cave, where some Egyptian rousette bats were sampled, is located in Rakai District where this most recent ONNV outbreak occurred ,17,18. HAnopheles [An. funestus was implicated in the 1996–1997 outbreak [An. funestus s.s.) is highly anthropophilic and a vector of human malaria in Africa [An. funestus can be variable and include other domestic animals [Anopheles funestus s.l. also comprises a large cryptic species complex that includes more exophilic species [Ae. aegypti, however this virus can also be transmitted by other anthrophilic Aedes species in forested habitats [Assuming exposure occurred through the bite of infected mosquitoes in or near QENP, transmission events could have occurred either at the cave roost location or at a nightly foraging site. ONNV is known to be transmitted by mosquitoes in the genus nopheles , and in outbreak . This mon Africa , althoug animals . Anophel species . The prihabitats ,39,40. IAnopheles mosquitoes were not commonly captured in the vicinity of Python cave during the time frame these bats were sampled. Mosquito collections from Maramagambo forest in 2009–2010 yielded over 50 species from 7 genera, of which a very small percentage were anophelines [Coquillettidia fuscopennata, and Culex decens group [Cq. fuscopennata, and Babanki (BBKV) and CHIKV from Cx. decens group mosquitoes [Alternatively, if the bats were exposed to ONNV in Maramagambo forest, it is unknown what potential mosquito vectors could be responsible, or when this exposure may have occurred. phelines . Mosquitns group . Both ofsquitoes . Therefosquitoes . NeitherPerimyotis subflavus) became infected with Japanese encephalitis virus (family: Flaviviridae) after consuming infected mosquitoes [The detection of such a high neutralizing antibody titer to ONNV in the little free-tailed bat was particularly surprising . The antsquitoes , setting75) in Egyptian rousette bats of 1:4 to 1:8, and not all infected bats developed neutralizing antibodies.Neutralizing antibody titers among the other bats were generally very low . AntibodSometime prior to 2009, ONNV and CHIKV appear to have been circulating in Western Uganda at high enough levels such that approximately 13% of Egyptian rousette bats in Maramagambo forest became exposed and developed detectable neutralizing antibody titers to ONNV and almost 1% to CHIKV. The epidemiological significance of this finding is unknown, but it seems unlikely that these bats could be natural reservoirs for either virus given the lack of seropositivity in the other populations tested. Whether or not Egyptian rousette bats support ONNV or CHIKV replication and could potentially serve as a zoonotic amplifying host during periods of elevated virus circulation is unknown. This evidence for exposure to both CHIKV and ONNV coupled with generic alphavirus antibody detection and prior detections of neutralizing antibodies to BBKV does indicate that Egyptian rousettes are exposed to multiple mosquito-borne alphaviruses. Moreover, a single seropositive little free-tailed bat indicates that this species may be susceptible to ONNV by oral exposure by feeding on infected mosquitoes, but further research is warranted.


Accurate tumour delineation on HP-13C-MRI is vital for quantitative assessment of the underlying tissue metabolism. However, there is no consensus on the optimum method for segmenting HP-13C-MRI, and whole-mount pathology (WMP) as the histopathological gold-standard is only available for surgical patients. Although proton MRI can be used for tumour delineation, this approach significantly underestimates tumour volume, and metabolic tumour segmentation based on HP-13C-MRI could provide an important functional metric of tumour volume. In this study, we quantified metabolism using HP-13C-MRI and segmentation approaches based on WMP maps, 1H-MRI-derived T2-weighted imaging (T2WI), and HP-13C-MRI-derived total carbon signal-to-noise ratio maps (TC-SNR) with an SNR threshold of 5.0. 13C-labelled pyruvate SNR, lactate SNR, TC-SNR, and the pyruvate-to-lactate exchange rate constant (kPL) were significantly higher when measured using the TC-SNR-guided approach, which also corresponded to a significantly higher tumour epithelial expression on RNAscope imaging of the enzyme catalysing pyruvate-to-lactate metabolism (lactate dehydrogenase (LDH)). However, linear regression and Bland-Altman analyses demonstrated a strong linear relationship between all three segmentation approaches, which correlated significantly with RNA-scope-derived epithelial LDH expression. These results suggest that standard-of-care T2WI and TC-SNR maps could be used as clinical reference tools for segmenting localised PCa on HP-13C-MRI in the absence of the WMP gold standard. The TC-SNR-guided approach could be used clinically to target biopsies towards highly glycolytic tumour areas and therefore to sample aggressive disease with higher precision.Hyperpolarised [1-• T2WI- and TC-SNR-guided segmentations can be used in all PCa patients and do not explicitly require WMP maps.• Agreement between the three segmentation approaches is biologically validated by their strong relationship with epithelial LDH mRNA expression.The TC-SNR-guided approach can potentially be used to identify occult disease on1H-MRI and target the most glycolytically active regions.• The online version contains supplementary material available at 10.1007/s00330-022-08929-7. However13C-MRI may be of clinical benefit. Conventional proton (1H) MRI is known to underestimate tumour diameter and volume up by up to 3-fold when compared to WMP [13C]lactate signal and the underlying tumour area on pathology is unknown, and could vary from representing only the metabolically active tumour core, through detecting labelled lactate beyond the anatomical boundaries of the lesion. In the former case, direct lesion segmentation on HP-13C-MRI may be sufficient for accurate tumour metabolic phenotyping based on assessing the glycolytic volume, which is known to harbour more aggressive disease [18F-fluorodeoxyglucose (18F-FDG) uptake [In localised disease, accurate tumour delineation is vital for definitive evaluation of the underlying tissue metabolism. However, whole-mount pathology (WMP) is only available following radical prostatectomy, making the histopathological gold standard for tumour delineation inaccessible for most patients in which HP-d to WMP , and may disease , 4, 6. T) uptake .13C-MRI segmentation approach that would best reflect underlying tumour metabolic characteristics. Tumour HP-13C-MRI-derived metabolic metrics and spatial LDH mRNA expression patterns were compared using three segmentation approaches: WMP, 1H-MRI, and HP-13C-MRI-derived total carbon signal-to-noise ratio maps (TC-SNR).This study aimed to identify a clinically applicable HP-13C-MRI, measuring pyruvate SNR, lactate SNR, TC-SNR, and the pyruvate-to-lactate exchange rate constant (kPL). Full imaging protocols for mpMRI and HP-13C-MRI have been described previously [This was a retrospective study of data acquired prospectively as part of an institutional review board–approved  study that included patients undergoing radical prostatectomy for biopsy-proven PCa . All pateviously . All pat2-weighted images (T2WI) in consensus by two radiologists  and a research fellow (N.S.) with 12, 13, and 4 years’ experience in prostate MRI, respectively. The segmentation was blinded to WMP maps, with PCa regions-of-interest (ROIs) defined by clinical interpretation as areas showing low T2WI signal intensity with corresponding restriction on diffusion-weighted imaging according to clinical guidelines [13C-MRI-derived TC-SNR maps, masked using a TC-SNR threshold of 5.0 according to the Rose criterion [13C-MRI metabolic maps .PCa foci were first outlined on digitised surgical haematoxylin-and-eosin (H&E) WMP maps by an experienced genitourinary pathologist (A.Y.W.)  prostatectomy tumour blocks using RNAscope in situ hybridisation technology, as detailed previously [LDHA and LDHB mRNA expression values were derived from WMP-, T2WI-, and TC-SNR-guided segmentations that were transposed onto co-registered RNAscope slides  and epithelial LDHA expression values were divided by the ROI surface area (mm2) to produce the total epithelial LDH and epithelial LDHA densities, respectively.Simultaneous detection of human s Figure . To acco13C-MRI-derived lactate SNR and kPL, and RNAscope-derived total epithelial LDH density and epithelial LDHA density, respectively, for the three segmentation approaches.Statistical analyses were conducted using GraphPad Prism . Data normality was assessed using the D’Agostino-Pearson test, with all pairwise comparisons conducted using the Wilcoxon matched-pairs signed-rank test. Spatial congruence between different segmentation approaches was assessed using the Dice similarity coefficient (DSC) . Simple The study comprised eight patients  with 11 MR-visible lesions, nine and two of which harboured Likert scores 5 and 4, respectively. Seven lesions were in the peripheral and four in the transition zones, with the median maximum axial tumour diameter measuring 16 mm . One tumour harboured Gleason score 3 + 3 = 6 disease, eight 3 + 4 = 7 disease, and two 4 + 3 = 7 disease.kPL showed no significant differences between the WMP- and T2WI-guided segmentation approaches .Linear regression analysis showed very strong positive correlations between lactate SNR from all three segmentation approaches Fig. . Bland-A13C-MRI-derived lactate SNR and RNAscope-derived total epithelial LDH density values extracted using the WMP-, T2WI-, and TC-SNR-guided segmentation approaches  coupled with a high spatial overlap between the two segmentation approaches, which was also reflected by the linear regression and Bland-Altman analyses. Therefore, T2WI alone is as reliable as the histopathological gold standard for HP-13C-MRI segmentation. Importantly, whilst TC-SNR-derived metabolic values were numerically different to those obtained using T2WI, the two segmentation approaches had strong linear relationship and agreement. In addition, lactate SNR measured using all three approaches showed strong positive correlations with the underlying tumour metabolic phenotype assessed by the epithelial mRNA density of LDH, the enzyme responsible for the imaged reaction [kPL and epithelial LDHA, which were shown to be closely related in a previous study [The observed differences between WMP- and T2WI-guided ROI surface areas are in line with the known underestimation of tumour measurements on 1H-MRI . Howeverreaction . This trus study . Clinicaus study .13C-MRI to navigate targeted sampling of 1H-MRI occult lesions was not assessed since all patients already had biopsy-proven disease. The study excluded 1H-MRI occult lesions due to their inability to be prospectively segmented using the T2WI-guided segmentation approach.This study has several limitations. The sample size was relatively small, albeit in line with similar publications in the field , 2, 6. T13C-MRI and targeting biopsies to sample the most glycolytically active tumour areas.In conclusion, the TC-SNR-guided segmentation approach generated smaller tumour regions with higher measures of metabolism in keeping with identification of the metabolic core on RNAscope. Although the T2WI approach generated larger regions with lower overall levels of metabolism, these were linearly correlated with the measures using the TC-SNR approach and those generated from the WMP gold standard. These findings support the use of TC-SNR maps for delineating localised PCa on HP-ESM 1(DOCX 2293 kb)


BRCA1 or BRCA2 pathologic variants, there is emerging evidence that patients with tumours harbouring pathologic variants in other HR genes may also benefit from PARPi therapy. The objective of this study is to assess the feasibility of primary-tumour testing by examining the concordance of variant detection between germline and tumour-variant status using a custom hereditary cancer gene panel (HCP). From April 2019 to November 2020, HCP variant testing was performed on 146 HGSC formalin-fixed, paraffin-embedded tissue samples using next-generation sequencing. Of those, 78 patients also underwent HCP germline testing using blood samples. A pathogenic variant was detected in 41.1% (60/146) of tumours tested, with 68.3% (41/60) having either a BRCA1 or BRCA2 variant (n = 36), or BRCA1/2 plus a second variant (n = 5), and 31.2% (19/60) carrying a pathogenic variant in another HCP gene. The overall variant rate among the paired germline and tumour samples was 43.6% (34/78), with the remaining 56% (44/78) having no pathogenic variant detected in the germline or tumour. The overall BRCA1/2 variant rate for paired samples was 33.3% (26/78), with germline variants detected in 11.5% (9/78). A non-BRCA1/2 germline variant in another HCP gene was detected in 9.0% (7/78). All germline variants were detected in the tumour, demonstrating 100% concordance. These data provide evidence supporting the feasibility of primary-tumour testing for detecting germline and somatic variants in HCP genes in patients with HGSC, which can be used to guide clinical decision-making, and may provide opportunity for improving patient triage and clinical genetic referral practices.Despite advances in treatment, prognosis for most patients with high-grade serous carcinoma (HGSC) remains poor. Genomic alterations in the homologous recombination (HR) pathway are used for cancer risk assessment and render tumours sensitive to platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi), which can be associated with more favourable outcomes. In addition to patients with tumours containing  BRCA1/2 germline testing in patients with high-grade epithelial ovarian carcinoma (EOC) is now considered the standard of care [BRCA-mutated high-grade EOC are eligible for poly (ADP-ribose) polymerase inhibitor (PARPi) maintenance therapy [ of care . In addi therapy . Several therapy ,6,7,8.BRCA1/2 variants can identify more patients who might be eligible for PARPi treatment [BRCA1/2 pathogenic variants occur in 22.6% of high-grade serous carcinomas (HGSCs), while somatic BRCA1/2 pathogenic variants have been shown in an additional 6–7% of HGSCs [BRCA1/2 pathogenic variant (germline or somatic) can benefit from PARPi therapy after the completion of front-line chemotherapy.Tumour testing can identify somatic variants, independent of germline status ,9. In coreatment ,12,13,14of HGSCs . Thus, aBRCA1/2 germline or somatic pathogenic variants in Canada [BRCA1/2 variant status between germline and somatic testing results showed 100 percent concordance, providing validation for the use of tumour testing to the determine potential utility of treatment, as well as hereditary cancer risk [In 2016, the use of PARPi for recurrent high-grade epithelial carcinoma of the ovary, fallopian tube, and peritoneum was approved for patients with n Canada . Moreoven Canada ,18. Reflcer risk .BRCA1/2 variants are currently considered the most clinically relevant, patients with variants in other HR pathway genes (either in germline or tumour) may also benefit from PARPi therapy [ATM, PALB2, TP53, and APC, in tumours from patients with HGSC [While germline and somatic  therapy . It is tith HGSC , no studThe objective of this study is to examine the concordance of hereditary cancer gene variants between germline and tumour testing in patients with HGSC using an institutional hereditary cancer gene panel (HCP) of 37 genes. We aim to demonstrate the feasibility of primary-tumour testing and assess the extent to which tumour testing can reliably capture germline pathogenic cancer gene variants.The study population included a retrospective cohort of all patients with HGSC at our institution from April 2019 to November 2020. Genetic tumour testing was previously performed on patient samples; this consisted of a clinically validated 37-gene Hereditary Cancer Panel (HCP), which enabled the simultaneous detection of both sequence and copy-number variations (CNV) of the target genes using next-generation sequencing (NGS). Germline testing was also previously reported and blinded prior to the assessment of tumour specimens. Tumour test results were compared with matched germline testing results, for those available. Access to funded genetic testing in Ontario is limited to patients meeting the criteria defined by government regulations, and therefore, only a subset of cases had previous germline testing results available.BRCA1/2 are assessed only. TP53 is included in the HCP, but was not assessed for the purposes of this study. The sensitivity detection of this custom NGS pipeline has been validated previously for minor allele detection levels at 2–5%, along with sub-exon-level CNV detection [The previously clinically validated 37-gene HCP examined all coding exons and 20 bp of flanking intronic sequences for the 37 genes , and wasetection .Three 20 μm sections from formalin-fixed paraffin-embedded (FFPE) tissue samples with adequate tumour cellularity were obtained for DNA extraction using a sterile protocol. Genomic DNA was isolated using the Invitrogen RecoverAll total nucleic acid isolation kit , according to the manufacturer’s protocol.Genomic DNA from each peripheral blood sample was isolated by standard protocols using the MagNA Pure system .NGS libraries were prepared as described previously ,23. BrieBRCA1 or BRCA2 was 30%. Sub-gene-level events were identified by a minimum of 50% deviation from the nomalised values of the remainder of the gene.Base coverage distribution reports were created using NextGene software  and processed through a normalisation algorithm described previously ,23. CNV Results are reported using descriptive statistics. For samples in which tumour and matched germline testing was available, variant identification was compared to determine concordance. In order to assess if tumour analysis could further direct germline assessment, type of variants by origin, tumour variant allelic fraction (VAF) based on origin, and sequence variant origin based on gene distribution were examined.n = 1) and duplicate specimens received (n = 3). Of the 146 FFPE HGSC tumour specimens tested, 41 (28%) carried a Tier I/II variant in either BRCA1 or BRCA2 (n = 36), or BRCA1/2 plus a second variant (n = 5). An additional 19 (13%) specimens carried one or multiple Tier I/II variants in the remaining HCP genes: APC (n = 1), ATM (n = 1), BARD1 (n = 1), BRIP1 (n = 1), CDKN2A (n = 2), MSH3 (n = 3), MUTYH (n = 4), NBN (n = 1), PALB2 (n = 1), PMS2 (n = 1), POLE (n = 2), PTEN (n = 3), RAD51C (n = 1), RAD51D (n = 2), and SDHB (n = 1). A complete list of variants identified are available in A total of 150 tumour samples from patients with HGSC were received for somatic tumour testing during the 19-month study period; however, 4 were removed from analysis due to an insufficient amount of DNA extracted  were labelled as germline-positive while ones without germline variants  represented somatic events. A total of 78 tumour specimens had matching germline assessments completed . Cases iBRCA1/2 tumour-positive cohort, 26 cases had matching germline assessments, with 9 (35%) being a germline event and 17 (65%) representing a somatic event. One of the somatic cases (Case 031) carried two BRCA1 variants and both were deemed somatic in origin  were of germline variant origin and 1/8 (12.5%) was a somatic event  than germline variants (n = 18), all types of sequence variants were represented across both variant origins. However, all large, whole-gene deletion events (n = 10) were observed as somatic in origin  showing a VAF greater than 40% A,B. SomaBRCA1 sequence variants accounted for 11/29 (38%) variants and 5/11 (45%) were of germline origin (BRCA2 sequence variants accounted for 6/29 (21%), and 4/6 (67%) were of germline origin (BRCA1/2) HCP genes, which accounted for 12/29 (41%) and 9/12 (75%), were of germline origin , rendering patients eligible for PARPi therapy.Our results indicate that EOC tumour testing can detect both In terms of somatic tumour testing, our results mirror those of Fumagalli et al., who found that 5/23 (21.7%) pathogenic/likely pathogenic variants were identified through tumour testing only and would not have been detected using germline testing alone .BRCA1/2 variants, one institution has seen improvements in the rate of genetic referral (12.88% versus 7.10%) and time to genetic counselling appointment (59 days versus 33 days) [In addition to expanding patient eligibility for access to targeted therapy, tumour testing, which can be performed reflexively as part of the routine pathology assessment at the time of diagnosis, has implications for hereditary cancer syndrome detection. In Canada, there are significant wait times for genetic counselling and reported referral rates can be extremely low (6.6%) . However33 days) . Similar33 days) ; howeverBRCA wild-type and HRD-negative tumours [BRCA1/2, other genes in the HR repair pathway, which may provide information for more complete ovarian cancer management, should also be analysed in tumour samples. Specifically, non-BRCA1/2 variants were identified in an additional 13% of tumour samples in our study .Our study shows that tumour testing can be performed to detect pathogenic variants in other HR pathway genes. Importantly, several studies have reported the efficacy of PARPi in patients with non-mutated BRCA high-grade EOC ,32,33,34 tumours ,35,36. ABRCA1/2 testing is mandated for the determination of PARPi therapy eligibility, and as such, tumour testing cannot supplant germline testing. As reported by others, and reiterated in our study, tumour testing for BRCA1/2 variants is a robust way to triage patients with BRCA1/2 variants, not only for PARPi therapy, but possibly also for genetic counselling [BRCA1/2 variants still have clinical relevance, until tumour testing is expanded, germline testing cannot be replaced. The American Society of Clinical Oncology (ASCO) guidelines recommend multigene panel germline testing for patients with ovarian carcinoma; this includes, at least, BRCA1, BRCA2, RAD51C, RAD51D, BRIP1, MLH1, MSH2, MSH6, PMS2, and PALB2, as these have been associated with the risk of inherited ovarian cancer [RAD51C, RAD51D, BRIP, MSH3, APC, MUTHY, and PALB2 were identified. In addition to the association between inherited ovarian cancer and RAD51, BRIP, and PALB2 pathogenic variants, MUTHY pathogenic variants confer an increased risk of ovarian cancer and may demonstrate resistance to platinum-based agents, much like tumours with mismatch repair deficiency [BRCA1/2 variants detected in tumours were of germline origin. In addition, expanding tumour testing may offer further information for tailoring targeted therapies.At this time, in Ontario, only nselling ,37. Recon cancer . In our ficiency . Our stuBRCA1/2 VAF to gain insights on variant origin has been previously examined [BRCA1/2 pathogenic variants was over 40% (44–94%), there was a wider range in VAF for variants of uncertain significance (5–90%) [Examining examined . While a (5–90%) . In our TP53 analysis was not reported on tumour samples as the pathogenesis of HGSC is driven by p53 dysfunction and TP53 pathologic variants are present in almost all HGSCs [TP53 germline variants were not reported. As one of the goals of this study is to provide support for tumour testing as a potential way to triage patients for genetic counselling and follow-up germline testing, including TP53 pathologic variants would flag high numbers of patients for further testing and would not be practical in our healthcare system. Second, this was a retrospective study of a 146-patient cohort; prospective studies with a larger patient cohort will provide further insight into the utility of tumour genetic testing for the purposes of therapy eligibility and genetic testing referrals. Studying FFPE tissue also comes with limitations due to the quality of the DNA specimens obtained, which can impact the detection of more complex variants, such as CNVs. The detection of all CNVs in this study were of somatic origin and mostly full-gene events. This could indicate the involvement of larger chromosomal rearrangements that were not assessed in this study. Without the assessment of any germline cases that carry a large CNV, additional studies are needed to determine the feasibility of primary detection in tumour specimens before the adaptation of this workflow. Alternatively, supplementary techniques, such as MLPA, could be performed in parallel to rule out any CNVs. Finally, the limit of detection for somatic variants in this study was 10%, but it has not been evaluated if those with a lower tumour heterogeneity could also benefit from targeted PARPi therapy.There are some general limitations to this study. First, the HCP utilised was a 37-gene panel; further expansion of the panel may detect other relevant variants. Moreover, although included in the HCP, ll HGSCs . TherefoBRCA1/2 variants allow for the identification of more patients who would be suitable for PARPi therapy. Additionally, tumour testing has the potential to enhance the efficiency of genetic testing. Because both germline and somatic cancer variants can be detected in tumours, tumour testing could act as an initial screen for eligibility for germline testing and could potentially replace initial germline testing. Lastly, for deceased patients with HGSC who did not undergo BRCA1/2 genetic testing, testing of archived tumour tissue to detect possible germline HCP variants could provide essential information to family members.The failure rate of tumour testing was low at our institution (0.67%), and reliable results were obtained from tumour samples following neoadjuvant chemotherapy (33.3% of cases). Proof of an efficient and robust tumour testing pathway has several advantages in clinical practice. First, by examining the molecular characteristics of a tumour at the time of diagnosis, timely and appropriate therapeutic decision-making can be made. In the context of HGSC, somatic BRCA1/2 and other HR genes in order to determine treatment eligibility and aid in triaging patients for germline testing and genetic counselling referrals. Further studies are required to examine the concordance of variant detection in tumour tissue, normal tissue, and germline to help further determine the validity of tissue testing directly as an initial screen for germline variant detection.This study provides support for reflex tumour testing with a comprehensive NGS panel that includes


The mortality and morbidity of meconium aspiration syndrome in neonates born with meconium-stained amniotic fluid in developing countries are still high. In Nepal, few studies have estimated the prevalence of meconium-stained amniotic fluid among newborns. The study aimed to find out the prevalence of neonates born through meconium-stained amniotic fluid among deliveries in a tertiary care centre.This descriptive cross-sectional study was conducted among neonates born in a tertiary care centre from November 2021 to March 2022. Neonates born during the study period with meconium-stained amniotic fluid were studied. Ethical approval was obtained from the Institutional Review Committee. Convenience sampling method was used. With permission from the Department of the Neonatal Intensive Care Unit, the data were collected and entered in a Microsoft Excel sheet. Point estimate and 95% Confidence Interval were calculated.Among 576 neonates, the prevalence of neonates born through meconium-stained amniotic fluid was 77 (13.37%) .The prevalence of neonates born through meconium-stained amniotic fluid was found to be similar to other studies done in similar settings. The common causes of respiratory distress are Transient Tachypnea of newborns, Hyaline Membrane Disease and Meconium Aspiration Syndrome.2The prevalence of respiratory distress in a tertiary care centre in Nepal was found to be 6.55% among the total live births and 30.83% among the admitted cases of the Neonatal Intensive Care Unit (NICU) with an estimated mortality of 4.50%.4If the foetal meconium passes into the amniotic fluid then it is called Meconium Stained Amniotic Fluid (MSAF) and usually occurs in approximately 15-20% of term pregnancies and 30-40% of the post-term pregnancies.The study aimed to find out the prevalence of neonates born through meconium-stained amniotic fluid among deliveries in a tertiary care centre.A descriptive cross-sectional study was conducted among neonates born in Kathmandu Medical College Teaching Hospital (KMCTH) from November 2021 to March 2022. Ethical approval was obtained from the Institutional Review Committee of KMCTH. Neonates born during the study period with meconium-stained amniotic fluid were studied. The inclusion criteria were all newborns born in KMCTH within the study period. The exclusion criteria were patients whose parents did not give consent for the study, neonates with birth injuries, neonates with congenital malformations, multiple gestations and preterm babies. Convenience sampling method was used. The sample size was calculated using the given formula:Where,n = minimum required sample size,Z = 1.96 at 95% Confidence Interval (CI),p = prevalence taken as 50% for maximum sample size calculationq = 1-pe = margin of error, 5%The minimum sample size calculated was 385. However, the final sample size taken was 576.Data were entered in Microsoft Excel sheet. Point estimate and 95% CI were calculated.Among 576 neonates, the prevalence of neonates born through meconium-stained amniotic fluid was 77 (13.37%) . Out of 77 meconium-stained liquor, 27 (35.06%) had thin meconium .Thirty-eight (49.35%) women were nulliparous among 77 women who gave birth to neonates who had meconium-stained amniotic fluid. Low-segment Caesarean Section was the mode of delivery in 50 (64.93%) women .Among 77 neonates born through meconium-stained amniotic fluid, 8 (10.39%) developed meconium aspiration syndrome .Premature rupture of membranes was seen in 6 (7.79%) mothers of neonates born through a meconium-stained amniotic fluid .11 Studies conducted in Nepal have revealed the prevalence of MSAF about 6.5%-14.6%,12 among all live deliveries, the prevalence of MAS among MSAF about 6.6% to 8.6%,12 and neonatal mortality from MAS about 5.4% to 11.3%.12 The study conducted in India found the prevalence of MAS among MSAF as 7.7% to 21.5%,6 and mortality among MAS as high as 13.8%.6 The most common complication of MAS is Respiratory distress and other morbidities were convulsions, sepsis, shock, hypoxic-ischemic encephalopathy, hyperbilirubinemia and hypoglycemia.13In our study, the prevalence of meconium-stained amniotic fluid among neonates was found in 77 (13.37%) deliveries. The incidence of MAS was high in Nepal with an overall incidence of about 2.0 per 1000 live births.12 among all live deliveries, the prevalence of MAS among MSAF about 6.6% to 8.6%,12 and neonatal mortality from MAS about 5.4% to 11.3%.12Several studies conducted in Nepal have revealed the prevalence of MSAF about 6.5%-14.6%,12 The cost of care and utilisation of critical care services are frequent targets of concern for the healthcare system in a resource-limited setting.MSAF is an important determinant for both maternal and neonatal morbidity and mortality. The complicated MSAF needs prolonged admission of neonates in the NICU.14 The perinatal morbidity and mortality related to MSAF can be decreased if major risk factors are recognized early and closely monitoring of the labour and careful decisions are made about the timing and mode of delivery.14Meconium-stained amniotic fluid not only poses risk to the newborn but also significantly increases the rate of maternal complications such as meconium-laden amniotic fluid embolism, intrapartum chorioamnionitis, puerperal endometritis, wound infection, increased risk of operative delivery and its complication.7 However, Meconium aspiration syndrome (MAS) is still an important cause of neonatal mortality and morbidity in neonatal intensive care units (NICUs) in developing countries.12With the improvement of obstetric practices and perinatal care, the incidence of MAS and other causes of Respiratory distress is decreasing in developed countries.This study is a single institution-based descriptive cross-sectional study, the results might not be completely generalizable in other settings. So, a study design with a higher level of evidence is recommended for future studies. There may be non-response data as data will be collected from the patient party. There may also be information bias and respondent bias.The prevalence of neonates born through meconium-stained amniotic fluid among deliveries was similar to other studies done in similar settings.


Pr, and K. Moreover, the concentration distribution shows diminishing behavior as we put the higher value of the Brownian motion number.Due to their unique microstructures, micropolar fluids have attracted enormous attention for their industrial applications, including convective heat and mass transfer polymer production and rigid and random cooling particles of metallic sheets. The thermodynamical demonstration is an integral asset for anticipating the ideal softening of heat transfer. This is because there is a decent connection between mathematical and scientific heat transfers through thermodynamic anticipated outcomes. A model is developed under the micropolar stream of a non-Newtonian (3rd grade) liquid in light of specific presumptions. Such a model is dealt with by summoning likeness answers for administering conditions. The acquired arrangement of nonlinear conditions is mathematically settled using the fourth-fifth order Runge-Kutta-Fehlberg strategy. The outcomes of recognized boundaries on liquid streams are investigated in subtleties through the sketched realistic images. Actual amounts like Nusselt number, Sherwood number, and skin-part coefficient are explored mathematically by tables. It is observed that the velocity distribution boosts for larger values of any of  Hayat et al.2 scrutinized rotating third-grade MHD fluid flow bounded between two permeable sheets. Farooq et al.3 probed mass and thermic transmission for third-grade fluid inside a vertically positioned flow configuration besides viscous dissipation. Sinha4 studied MHD third-grade fluid flow inside a pipe having stretched and porous walls. Okoya5 examined the heat-exchange properties of an exothermic reactive third-grade fluid across a circular passage by considering the Reynolds viscosity model and its uses in the processing industries. Hayat et al.6 explored MHD third-grade fluid flow bounded with convective heated and stretched surfaces. Magnetohydrodynamic stagnation point flow for non-Newtonian (third-grade) fluid caused by the non-linearly stretchy surface is examined by Hayat et al.7. Interested readers can see some recent work on third-grade fluids here11.Many researchers around the globe are showing a keen interest in learning more about the non-Newtonian fluid flow. The motivation for studying these fluids is their potential usage in industries and technology. These fluids have paramount prominence in material processing, bioengineering, geophysics, oil reservoir engineering, chemical, nuclear, and many other fields. Many materials of our daily life use, such as apple sauce, mud, paints, shampoos, soaps, ice cream, condensed milk, polymeric liquids, low shear rate blood, pasta, oils, etc., show a highly complex nature and are taken as non-Newtonian liquids. Rheological physical attributes of a non-Newtonian fluid are impossible to explore by using the simple Navier-Stokes theory (unlike for viscid fluids). The third-grade fluid is one of the categories of differential type fluids. It describes shear thinning and thickening properties. Abbasbandy et al.12 was the pioneer who studied the flow of micropolar fluids. Physical examples of micropolar fluids can be seen in ferrofluids, blood flows, bubbly liquids, liquid crystals, and so on, all of them containing intrinsic polarities. Eringen’s hypothesis12 proclaims that any fluid formed from rigid, arbitrarily oriented, or spherical particles with particular micro-rotations is called the micropolar fluid. Guram and Smith13 studied flow subject to stagnation point for a micropolar liquid model. The flow model of a micropolar liquid caused by a heat source over an axisymmetric and rotating sheet is probed by Gorla and Takhar14. By considering combined convective conditions, Gorla et al.15 presented an axisymmetric stagnation point stream through a perpendicular standing cylinder for micropolar fluid. MHD effects on micropolar liquid stream across a continually moving plate were investigated by Sadeek16. Eldahab et al.17 probed the influence of radiations on thermal transmission for a micropolar liquid stream across a flat sheet in a permeable medium. Mohamed and Abo-Dahab18 deliberated the consequences of thermal radiations and chemical reactions on mass and thermal transport for micropolar fluid in penetrable media. Investigation for the micropolar fluid flow due to permeable stretched sheet also consequent thermal transmission is done by Turkyilmazoglu19. The darcy-Forchheimer flow of 3D micropolar fluid along horizontal parallel sheets in the revolving system in a penetrable medium is analyzed by Khan et al.20. For an in-depth investigation of micropolar fluid flow, we refer the curious reader to some recent studies25.Eringen26 introduced the investigation of stream subject to an extended sheet. Brady and Acrivos27 concentrated on the liquid stream in an extended channel. Specialists observed that an answer for a particular worth of Reynolds number exists for a 2D stream. Ahmad et al.28 performed the computational investigation of an unstable (3-D) chemically reacting MHD flow of Maxwell fluid. Ahmad et al.29 used the Cattaneo-Christov thermal influx model to probe the hybrid Micropolar nanoliquid stream with triple stratification. Many researchers have investigated heat and thermal investigation of fluids over stretched surfaces36. Khan et al.37 explored the MHD stream of second-grade fluid with multiple slip constraints. The non-Newtonian fluid has been widely discussed by42.Over the decade, stream due to the extendable sheet has acquired astonishing significance among scientists due to its modern design execution. Not many of these applications incorporate hot rolling, paper production, glass blowing, polymer and metal expulsion, and precious stone development. Crane49, a third-grade micropolar liquid stream subject to a stretching sheet is investigated here. The primary purpose of this extensive study is to improve thermal transportation subject to the existence of micro-rotations of tiny nanoparticles. The numerical plan is made in the approaching segment by utilizing liquid stream presumptions. With the utilization of likeness changes, an arrangement of PDEs is decreased into a non-straight arrangement of ODEs. The mathematical strategy RK45 is used to settle the acquired framework systematically. The influence of some pertinent parameters is spotlighted on each temperature field The above-cited literature review shows that little attention is paid to the study considering the effects of third-grade micropolar fluid flow over an expanding surface. Motivated by the numerous uses of non-Newtonian fluids and nanofluidsentclass1pt{minimamentclasspt{minimaentclass1pt{minimax-axis is supposed along the continuously expanded surface while y-axis is taken perpendicular to this assumed surface in upward direction to the fluid , v and y are v and u are the parts of velocity in y and x directions respectively. N being Part of microrotation vector, orthogonal to the considered plane, and T for temperature, j is microinertia density, k is the vortex viscosity, and Steady incompressible and 2D stream of micropolar liquid over an extendable sheet is to be investigated in this communication. The See Fig. . For theAlso, suppose thatThe Rosseland radiative heat flux is Equation  is now tDefining the similarity variables   ting Eq.  in Eq. , an, an\docuting Eq.  are tranWith the altered BCs as:B is microinertia density parameter,Ec, Pr, Le are Eckert, Prandtl, Hartmann, and Lewis number respectively, Nt is Thermophoresis parameter, Nb is Brownian motion parameter, K the micropolar parameter, Rd the radiation parameter, Here involved dimensionless variables are given asThe local Sherwood number The above expressions in the form of similarity variables are:The PDEs system describing the above model is transmuted into an ODEs system by using suitable similarity variables. These ODEs system is handled by Runge-Kutta 4th order (RK-4) numerical method. Generally RK-4 method is most familiar. It is easy to implement, self-starting, and very stable method. The numerical solution for the problem is done by taking fixed values of similar variables In this research, the micropolar 3rd grade fluid flow past an exponentially stretchable sheet is studied. By understanding fluid flow assumptions, the mathematical model is constructed. Introducing the relevant similarity transformations, the system of PDEs is changed into ODEs. Equations. (12)–(15) along with the BC (16) is numerically solved by use of MATLAB. MATLAB software utilizes Runge-Kutta-Fehlberg fourth-fifth order shooting technique to give numerical solutions for BVP. The charactoristics of pertinent parameters involved on the Figure Pr. Perceptions are that thermal boundary layer thickness appears to decline while the rising upsides of Pr are utilized. As a result, when the Prandtl number ascents, pace of thermal conductivity gets higher. In this way, with more noteworthy Pr, heat will disperse more rapidly from the sheet. As it’s undeniably true that liquids having prevalent Prandtl number Pr will have lesser worth of thermal conduction. Therefore, Prandtl number is utilized to augment the cooling behavior in the flows. Heat exchange for different values of Radiation parameter Rd is presented in Fig. Rd and temperature distribution. It is concluded that with upper values of Rd results in enhancement of temperature distribution. In fact, asending value of parameter Rd means more heat is transferred to the fluid, that takes temperature distribution to go upward. Figure K on K. Figure Ec values. By boosting Ec, an elevation is seen in the temperature distribution. It transfers kinematic energy into inner energy by overcoming viscous fluid tension and converting it to heat. Higher values of the Eckert number enhance the system’s kinetic energy, which raises the temperature. As a result, increased viscous heat dissipation causes both growing heat and rising temperature.Figure Le on Le implies the lower Nb on the concentration field. A rise in Brownian motion variable gives a fall in Nt on B is seen on micropolar profile. The behavior of B.The role of the mentclasspt{minimax-direction has a significant impact on the magnitude of primary skin friction coefficient for all axial coordinate values.In Table In Table Pr, K, Ec, Rd, Nt is used while Nusselt number shows a decline for higher input of Rd improve convective heat transmission which ultimately raises the average Nusselt number.It is seen that Nusselt number gives a rise in its values as a larger value of Le and thermophoretic parameter Nt and decrement for higher value of Brownian motion number Nb.In Table Velocity distribution Rd, and Eckert number Ec, while, the opposite behavior for Pr, and K.Temperature distribution Nb and Le. On the contrary, this has the opposite behavior for Nt.Concentration distribution B.Micropolar distribution Skin-friction coefficient goes on increasing for higher values of parameters Pr, K, Ec, Rd, Nt are used, while Nusselt number depicts a decline for higher input of parameter Nusselt number shows an increasing behavior as larger values of parameters Le and Nt and decrement for the higher value of parameter Nb.Sherwood number shows increment for bigger values of parameters Third-grade micropolar fluid flow is analyzed in this research article. An ODEs system is generated from the PDEs system by a similarity transformation and then analytically solved by the fourth-fifth order Runge-Kutta-Fehlberg strategy. Several graphs are presented for the temperature, velocity, concentration, and micropolar fields to observe the impact of different parameters on them. The impact of various parameters on Nusselt number, skin-friction coefficient, and Sherwood number is also concluded. The main findings are