More than 30 million children worldwide suffer from moderate acute malnutrition(MAM). Current treatments have limited effectiveness and much remains unknown aboutpathogenesis. Children with MAM exhibit perturbed development of their gutmicrobiota.Slum-dwelling Bangladeshi children, aged 12 to 18 months, with moderate acutemalnutrition (n=124) received a microbiota-directed complementary food (MDCF-2) or anexisting ready-to-use supplementary food (RUSF), twice daily for three months followedby a 1-month period of monitoring. We obtained weight-for-length, weight-for-age, andlength-for-age Z-scores and mid-upper arm circumference at baseline and fortnightly,through four months. We compared the rate of change of these related phenotypes betweenbaseline and three months, and between baseline and four months. We also measured levelsof 4,977 proteins in plasma plus 209 bacterial taxa in fecal samples.118 children completed the intervention (n=59/arm). The rate of change inweight-for-length Z-score (β-WLZ), weight-for-age Z-score, and mid upper armcircumference is consistent with a benefit of MDCF-2 on growth over the course of thestudy including the one-month follow-up. Receipt of MDCF-2 was linked to the magnitudeof change in levels of 70 β-WLZ-positively correlated plasma proteins includingmediators of bone growth, neurodevelopment and inflammation  and the abundances of 23 WLZ-associated bacterial taxa.ClinicalTrials.gov identifier:NCT04015999)These findings provide support for further clinical investigation of MDCF-2 asa dietary supplement for young children with MAM and provide insight into mechanisms bywhich this targeted manipulation of microbiota components may be linked to growth.(Supported by the Bill and Melinda Gates Foundation and the NIH;  Childhood undernutrition is a global health challenge that produces impaired ponderaland linear growth (wasting and stunting), immune and metabolic dysfunction, altered centralnervous system (CNS) development as well as other abnormalities,2. AcuteWe previously identified a network (‘ecogroup’) of 15 bacterial taxawhose covarying representation describes normal gut microbial community development during thefirst 2 years of postnatal life in healthy members of birth cohorts from several low- andmiddle-income countries. CompariThis study was approved by the Ethical Review Committee at the icddr,b andconducted in Mirpur between November 2018 and December 2019. Parents/guardians of allstudy participants provided written informed consent.Male and female children with MAM between 12- and 18-months-old who satisfiedthe inclusion/exclusion criteria (n=124) were randomized to receive MDCF-2 or RUSF , weight-for-age z-score (WAZ), mid-upper-arm circumference (MUAC), length-for-agez-score (LAZ), morbidity, plasma proteomic profile, and gut microbiota configuration.Changes in ponderal growth between treatment arms were compared using linearmixed-effects models that included a random-effect coefficient to control for differencesin characteristics between individuals; other anthropometric measures were assessed in asimilar fashion. Analyses of food-frequency questionnaire and morbidity data wereperformed using generalized linear mixed-effects models. Because we tested the effects ofthe supplements on four measures of growth and did not correct for multiple testing, wehave provided confidence intervals for each outcome.Changes in plasma protein abundances were analyzed using an Empirical Bayeslinear model framework [limma] and genChildren with MAM, 15.4±2.0 (mean±SD) months-of-age, living inMirpur were enrolled in a 3-month randomized study of twice-daily dietary supplementationwith either MDCF-2 (n=61) or RUSF (n=62) . Fifty-nine children in each armcompleted the 3-month intervention and 1-month follow-up. Five did not complete the study,due to the family moving or withdrawal of consent .At enrollment, anthropometric and socio-demographic characteristics did notdiffer between children in the two arms  yielded β-indices of 0.011  forWLZ, 0.008  for WAZ, 0.003  for MUAC, and−0.001  for LAZ   higher or lowerlevels after the 3-month MDCF-2 supplementation, in contrast with 82 proteins that showedsignificant alterations after RUSF treatment . Proteins that showed increasesafter 3 months of supplementation with MDCF-2 were significantly enriched for the 70positively β-WLZ-associated proteins (GSEA p<0.001), in contrast with theproteins that showed increases after RUSF supplementation .Comparing the two treatments revealed that proteins whose levels increased more withMDCF-2 were significantly enriched for WLZ-associated proteins  (n=15 children/group) revealed that those in the upperquartile started off more wasted, were more deplete of mediators of bone growth andneurodevelopment and had higher levels of pro-inflammatory proteins compared to those inthe lower quartile. By the end of MDCF-2 supplementation, children in the upper quartilemanifested the largest increases in mediators of bone growth and CNS development and thelargest decreases in effectors of inflammation . Together, these results provide evidence that mediators of bonegrowth, neurodevelopment and inflammation distinguish the effects of themicrobiota-directed nutritional intervention from that of RUSF.To determine the effects of supplementation on gut microbiota, fecal sampleswere obtained every 10 days during the first month of the intervention and monthlythereafter. Quantitative PCR assays revealed no statistically significant differencesbetween treatments in the representation of 23 bacterial, viral and protozoanenteropathogens (Table S12). A more comprehensive analysis was obtained by identifyingbacterial taxa through sequencing of PCR amplicons generated from variable region 4 of 16SrDNA genes present in fecal biospecimens . We then used linear mixed-effectsmodels to determine the relationships between the abundances of bacterial taxa  andWLZ in each participant.Bifidobacterium sp.  and Escherichia coli (ASV_3) ; 21 were positively associated while two werenegatively associated ( (ASV_3)  are memb (ASV_3) . In thisum longum. Five ofum longum share tatory taxa. An analP. copri and F. prausnitzii,while the Bifidobacterium sp. (likely B. longum)exhibited the greatest decrease. Additionally, compared to (unsupplemented) children fromMirpur with consistently healthy growth phenotypes (WLZ ≥−1) and normallydeveloping microbiota, MDCF-2 produced a significant enhancement in the rate of change inabundances of ASVs assigned to members of Prevotella (includingP. copri), F. prausnitzii, Olsenella,and Bifidobacterium (B. longum) – i.e. thoseamong the most highly positively and negatively correlated with WLZ. In contrast, RUSF didnot elicit significant differences in the rate of change in abundance of any of the 23WLZ-correlated ASVs . One month after cessation ofsupplementation, levels of a majority of MDCF-2-responsive, WLZ-associated ASVs had begunto fall at the same time that WLZ was diminishing .The abundances of WLZ-associated taxa were significantly greater in the gutmicrobiota of children whose diets were supplemented by MDCF-2 than in those of childrenwhose diets were supplemented by RUSF  against an existingsupplementary food (RUSF) on four measures of growth in 12–18-month-old Bangladeshichildren with MAM. The rate of changes we observed in WLZ score, WAZ score and MUAC, but notLAZ score, support a benefit of MDCF-2 on growth despite its lower caloric density comparedto RUSF. We observed larger changes in plasma protein mediators of bone growth,neurodevelopment and inflammation and more complete repair of the gut microbiota of childrenwho received MDCF-2 compared with those who received RUSF.We did not attempt to test the effects of MDCF-2 and RUSF on body composition(changes in fat-versus lean-mass). Chronic undernutrition in early life induces metabolicreprogramming that may enable a child to more efficiently capture and store energy as fatduring periods of nutrient scarcity. WhileaStudies conducted in children with MAM in Malawi and Ethiopia indicate thatincreases in ‘fat-free’ mass accretion in the first two years of life areassociated with better cognitive and motor development,34. ChilBifidobacterium longum-dominated community during exclusive/predominantbreastfeeding, to one in which Prevotella copri becomes a major componentduring weaning is the transition from ag weaning. While BLarger trials performed in disparate geographies are needed to further assess theefficacy of this therapeutic approach for treating childhood undernutrition. The plasma andmicrobiota biomarkers identified in the present study should help enable bettercharacterization (and stratification) of participants in future interventions.Click here for additional data file.Click here for additional data file.


E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone  showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and  Glioblastoma multiforme (GBM) is the most frequent form of primary malignant brain tumor in adults , and it Highly efficient and tumor-selective gene transfer achieved by RRV enables efficacious prodrug activator gene therapy, which involves expression of enzymes that can convert precursor prodrugs into active chemotherapeutic drugs within the transduced tumor cells. This essentially forces the tumor to self-generate the chemotherapy drug from within. However, due to the relatively restricted payload capacity of RRV, which can only accommodate approximately 1.3-kb of additional transgene sequence inserted into the full-length replication-competent viral genome, it has generally been difficult to insert more than a single therapeutic gene per vector. For prodrug activator gene therapy, this is the equivalent of single-agent chemotherapy generated directly within the tumor.Analogously, since combination chemotherapy with multiple drugs is generally more efficacious than single-agent chemotherapy, there may be potential to achieve greater efficacy if multiple RRV encoding different prodrug activator genes could be combined. However, in this case the retrovirus envelope sequence in different vectors may need to be varied, so that these vectors do not compete for the same cell surface receptor, a phenomenon which in retrovirology is classically described as “receptor interference”. In particular, MLV-RRV currently in clinical development expresses the MLV 4070A strain amphotropic envelope, which binds the ubiquitous inorganic phosphate transporter, PiT-2 . In contE. coli nitroreductase (NTR)  fluoroethyl-L-tyrosine (L-[18F] FET) microPET was superior to [18F] FDG microPET for the monitoring of brain tumor due to the low uptake of L-[18F] FET in normal brain [18F] FET revealed sizable RG-2 gliomas at the tumor implantation site in the group treated with GS4-NTR alone. A clear difference between tumor-implanted and normal brains was observed. In contrast, rats treated with GS4-NTR plus CB1954 showed smaller tumors remaining at the tumor implantation site, demonstrating the therapeutic benefit achieved by GALV-based RRV-mediated prodrug activator gene therapy.Our previous study reported that al brain . MicroPEPersistent nonlytic infection of tumor cells by RRV facilitates the widespread seeding of prodrug activator genes, thereby allowing synchronized cell killing triggered by prodrug administration. Using GALV-based RRV expressing the CD and NTR suicide genes followed by the administration of the prodrugs 5-FC and CB1954, we have achieved highly efficient killing of glioma cells both in culture and in vivo, resulting in significantly prolonged survival in an immune-competent intracerebral glioma model.E. coli NTR isoform B (NfsB) gene cloned into the GS4-NTR vector expresses an enzyme that actively converts CB1954 to a 4-hydroxylamino derivative that is subsequently converted into a potent cytotoxic bifunctional alkylating agent capable of cross-linking DNA and therefore achieving cell-cycle-independent killing of both actively proliferating and non-proliferating cells  FET in the rats.L-[eviously . Static t-test was used for statistical analysis of in vitro cytotoxicity results. Kaplan-Meier analysis and log-rank tests were used to evaluate survival. All analyses were conducted using SAS software .Student’s


Background: Fatty liver, a major health problem worldwide, is the earliest pathological change in the progression of alcohol-associated (AFL) and non-alcoholic fatty liver disease (NAFL). Though the causes of AFL and NAFL differ, both share similar histological and some common pathophysiological characteristics. In this study, we sought to examine mechanisms responsible for lipid dynamics in liver and adipose tissue in the setting of AFL and NAFL in response to 48 h of fasting.Methods: Male rats were fed Lieber-DeCarli liquid control or alcohol-containing diet (AFL model), chow or high-fat pellet diet (NAFL model). After 6–8 weeks of feeding, half of the rats from each group were fasted for 48 h while the other half remained on their respective diets. Following sacrifice, blood, adipose, and the liver were collected for analysis.Results: Though rats fed AFL and NAFL diets both showed fatty liver, the physiological mechanisms involved in the development of each was different. Here, we show that increased hepatic de novo fatty acid synthesis, increased uptake of adipose-derived free fatty acids, and impaired triglyceride breakdown contribute to the development of AFL. In the case of NAFL, however, increased dietary fatty acid uptake is the major contributor to hepatic steatosis. Likewise, the response to starvation in the two fatty liver disease models also varied. While there was a decrease in hepatic steatosis after fasting in ethanol-fed rats, the control, chow and high-fat diet-fed rats showed higher levels of hepatic steatosis than pair-fed counterparts. This diverse response was a result of increased adipose lipolysis in all experimental groups except fasted ethanol-fed rats.Conclusion: Even though AFL and NAFL are nearly histologically indistinguishable, the physiological mechanisms that cause hepatic fat accumulation are different as are their responses to starvation. Fatty liver is a major health problem both in the United States and around the globe. It is the earliest and most common response to excessive ethanol consumption or over-consumption of a high-fat/high-sugar diet . It is cIn the body, liver and adipose tissue play prominent roles in maintaining energy homeostasis in both fed and fasting states. Specifically, hepatocytes play an important role in glucose homeostasis by storing or producing glucose in response to a fed or fasted state . AdiposeExtensive studies conducted in our laboratory as well as others have shown that multiple pathways in adipose-liver crosstalk contribute to the development and progression of liver disease in both AFL and NAFL . It is aAntibodies and reagents were purchased from the following companies: Ethanol was purchased from Pharmaco-AAPER . IRDye infrared secondary antibodies (Abs) and blocking buffer were from LI-COR Biosciences . Antibodies for adipose triacylglycerol lipase (ATGL), hormone sensitive lipase (HSL), and phosphorylated HSL (pHSL) were purchased from Cell Signaling , phosphorylated pATGL was obtained from Abcam , and perilipin 2 (PLIN2) antibody was from Fitzgerald . All other chemicals were obtained from Sigma Chemical Co.  unless stated otherwise.ad libitum either high-fat pellet diet  or maintained on standard chow pellet diet . NAFL and chow groups were allowed ad libitum access to their pellet diets and water  and weighed. Tissue sections were placed in 10% formalin for sectioning and remaining tissue was frozen in liquid nitrogen and stored at −80°C until further use. All animals received humane care in accordance with the guidelines established by the American Association for the Accreditation of Laboratory Animal Care. All protocols were approved by the Institutional Animal Care and Use Committee at the VA NWIHCS Research Service.Male Wistar rats (175–200 g) were purchased from Charles River Laboratories . For the AFL model, rats were weight-matched and pair-fed Lieber-DeCarli ethanol liquid diet  or control diet  for 6–8 weeks as described previously . Rats wend water . Rats innd water . These rParaffin-embedded liver and epididymal white adipose tissue was deparaffinized and stained with hematoxylin followed by counter-staining with eosin.Serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and cholesterol were measured by a Vetscan chemistry analyzer  using a Mammalian Liver Profile reagent rotor (#89126-004). Serum non-esterified fatty acids (NEFA) were measured in an enzymatic colorimetric assay using a HR Series NEFA-HR2 kit . Serum triglycerides (TG) were quantified using a colorimetric diagnostic kit (#TR22421) from Thermo Fisher Scientific .Measurement of liver TG were performed by lipid extraction of frozen liver pieces by the Folch procedure . AliquotImmunohistochemical staining for PLIN2, a lipid droplet protein, was conducted as described previously . ParaffiLiver homogenates were prepared in TE buffer containing protease inhibitor cocktail . Liver samples were subjected to SDS-PAGE, followed by blotting of proteins to nitrocellulose membranes. The proteins were detected with specific primary antibodies and fluorescently labeled secondary antibodies. The proteins were visualized and quantified using the Odyssey Infrared Imager and associated software .RNA was isolated from liver and epididymal adipose tissue using the PureLink RNA Mini Kit  and reverse-transcribed using the High Capacity cDNA Transcription Kit . Messenger RNAs were measured using iTaq Universal SYBR Green Supermix  and primers from Integrated DNA Technologies . PPARγ: Sense 5′- GAGATCCTCCTGTTGACCCAG-3′; Antisense 5′-CC ACAGAGCTGATTCCGAAGT-3′, PPARα: Sense 5′-GTCCT CTGGTTGTCCCCTTG-3′; Antisense 5′- GTCAGTTCACAGG GAAGGCA-3′, FAS: Sense 5′- TCCCAGGTCTTGCCGTGC-3′; Antisense 5′-GCGGATGCCTAGGATGTGTGC-3′, FATP2: Sense 5′- AGTACATCG GTGAACTGCTTCGGT-3′; Antisense 5′-TGCCTTCAGTGGAAGCGTAGAACT-3′, CD36: Sense 5′-AACCCAGAGGAAGTGGCAAAG-3′; Antisense 5′-GACAG TGAAGGCTCAAAGATGG-3′. Samples were analyzed using the 7500 Real Time PCR System . We used rat-specific primers from Applied Biosystems for mRNA analysis of monocyte chemoattractant protein-1  and interleukin 1 beta . The ΔΔCt method was used to determine the fold change of each transcript using β-actin or 36B4 mRNA for normalization.t-test. p-values of <0.05 were considered significant. For statistical analyses, data from ethanol-fed rats and their fasted cohorts were compared with pair-fed control rats. In the NAFL group, HFD-fed rats and their fasted cohorts were compared with chow-fed rats.Results are expressed as mean values ± SEM. Comparison between fed and fasted animals in AFL and NAFL groups were analyzed using the Student’s n = 8; data expressed as mean ± SEM), we did see significantly higher body weights in HFD-fed rats compared to chow-fed rats  and alkaline phosphatase (ALP) to investigate the effect of 48 h fasting on hepatic injury. As expected, activity of serum ALT and ALP increased in both ethanol and HFD-fed rats compared with pair-fed control and chow-fed rats, respectively . WhereasWe also measured serum lipid content in all experimental groups. Serum NEFA, TG, and total cholesterol levels were higher in ethanol and HFD-fed rats compared with their respective controls . As expeWe collected livers from fed and fasted rats to examine fat content by both histological and biochemical assessments. The histopathological evaluations of the formalin-fixed H&E stained liver sections were consistent with previous reports in that both alcohol and HFD-fed rats showed higher hepatic fat accumulation with evidence of both micro and macrovesicular steatosis as compared with their respective controls . Since wWe measured mRNA that encodes fatty acid synthase (FAS), which catalyzes fatty acid synthesis. Because we observed elevated TG in the livers of alcohol and HFD-fed rats, we expected higher levels of FAS mRNA. While ethanol-fed rats showed higher FAS mRNA expression than their pair-fed controls, HFD-fed rats showed lower FAS mRNA levels than respective controls, suggesting that lipogenic pathways are upregulated during the development of AFL , but notSince fat accumulation represents either increased fat synthesis and/or lower fat catabolism, we measured the activities of two main lipases, adipose triacylglycerol lipase (ATGL) and hormone sensitive lipase (HSL), which catalyzes triglyceride breakdown. Western blot analysis  revealedSeveral studies have reported that adipose-derived free fatty acids are taken up by the liver and esterified to form TG, which can be utilized either for energy production, gluconeogenesis or stored in lipid droplets during fatty liver development. Several proteins are responsible for hepatic fatty acid uptake/transport, including fatty acid transport protein 2  and cluster of differentiation 36 . As expected, we observed increased expression of hepatic FATP2 and CD36 in ethanol and HFD-fed rats compared with pair-fed control and chow-fed rats, respectively . Except Since it has been reported that fatty liver can promote inflammation , which iWe collected the epididymal white adipose tissue (eWAT) from fed and fasted animals to examine adipocyte size, which is representative of fat content. The histological evaluations of the formalin-fixed H&E stained eWAT revealed a decrease in the adipocyte size of ethanol-fed rats compared with controls, which is consistent with previous reports . HFD-fedFatty liver arising from either excessive alcohol consumption or over-consumption of a high-fat/high-sugar diet have similar phenotypes of hepatic lipid accumulation In our previous studies, we reported that despite showing similar phenotypic characteristics of accumulation of hepatic lipids and increased oxidative stress in livers of rats with AFL or NAFL, though there are mechanistic differences including impairment in membrane trafficking, which is observed only in AFL development . Here, wde novo lipogenesis and adipose-derived fatty acid uptake and esterification as well as decreased fatty acid oxidation and VLDL secretion . We observed increased expression of FAS mRNA in the livers of ethanol-fed rats compared with pair-fed controls, suggesting that increased de novo fatty acid synthesis contributes to ethanol-induced hepatic fat accumulation. Further, alcohol metabolism-induced increases in the NADH/NAD ratio could promote de novo fatty acid synthesis  abstinence-related recovery of the liver and adipose tissue, (ii) an increase in liver fatty acid oxidation as evident by increased PPARα, and (iii) the absence of further increases in the supply of adipose-derived NEFA to the liver because of an inadequate starvation-induced lipolytic stimulus as observed in pair-fed control rats.It is noteworthy that there was no further change in the hepatic p-ATGL (active form) between fed and fasted ethanol rats, and that p-HSL (active form) decreased in fasted ethanol-fed rats compared with fed-counterparts. This decrease in active HSL after fasting likely contributes to the residual fat accumulation due to deceleration of lipolysis in the livers of fasted ethanol-fed rats.In congruence with previous reports, ethanol-fed rats showed decreased epididymal white adipose tissue (eWAT) weight and adipocyte size while both were significantly elevated in HFD-fed rats compared with their respective controls. In correlation with adipocyte size, we observed increased HSL enzyme activity and decreased expression of PPARγ, which induces adipose differentiation and increases its fat storing capacity in the eWAT of ethanol-fed rats compared with control. These ethanol-induced changes in eWAT promote adipose lipolysis, causing a rise in adipose-derived serum NEFA levels as reported in our recent study . FastingWe observed that the dietary fat mainly contributed to the higher serum NEFA levels in HFD-fed rats compared with chow-fed rats. However, the increased HSL activation seen in eWAT of HFD-fed rats may have also contributed to the rise in NEFA. Fasting for 48 h induced similar responses in the eWAT of chow and HFD-fed rats and caused a further increase in HSL activation to promote eWAT lipolysis (as judged by the decrease in adipocyte size compared to fed-counterparts) ultimately leading to elevated adipose-derived serum NEFA levels.Increased fat accumulation makes the liver susceptible to inflammatory mediators, thereby, promoting the development of advanced liver injury. We observed elevated levels of the chemokine MCP-1 in livers of ethanol- and HFD-fed rat which is consistent with previous reports . Howeverde novo fatty acid synthesis, higher uptake of adipose-derived NEFA and impaired fatty acid breakdown contribute to the development of AFL. In the development of NAFL, however, increased dietary fatty acid uptake by the liver plays a major role. Likewise, the response of each model to fasting is also diverse. While hepatic steatosis decreased after fasting in ethanol-fed rats, animals with NAFL had increased hepatic steatosis compared with their fed-counterparts. This rise in hepatic steatosis after 48 h fasting of HFD-fed rats was likely due to increased delivery to the liver and enhanced hepatic uptake and esterification of adipose-derived NEFA combined with reduced TG lipolysis. The decrease in hepatic steatosis in AFL rats after 48 h fasting likely resulted from a combined effect of abstinence-related recovery, an increase in fatty acid oxidation and the absence of any further increase in the supply of adipose-derived NEFA to the liver.In summary, although AFL and NAFL feeding models showed similar histology, the physiological mechanisms in fatty liver development in each are different. Increased hepatic The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at the VA NWIHCS Research Service.KR: funding acquisition, conception, design of the study, data analysis and interpretation, and writing the original draft. CJ: performing the experiments, data acquisition, and data analysis. PT, JK, ES, and SS: aided in performing the experiments. GT: histological evaluation. TD: helped with interpretation of the data and editing the manuscript. MM: funding acquisition. KK: funding acquisition, data interpretation, and review and editing the manuscript. CC: funding acquisition, supervision of the project, and review and editing the manuscript. All authors contributed to the article and approved the submitted version.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.


Cancer patients are at increased risk of novel coronavirus disease 2019 (COVID-19). Currently, surgeries for cancer patients with COVID-19 are generally suggested to be properly delayed.We presented a 69-year-old Chinese female colon cancer patient with COVID-19, the first case accepted the surgical treatment during the pandemic in China. The patient developed a fever on January 28, 2020. After treatments with Ceftriaxone and Abidol, her fever was not moderated yet. A repeat chest computed tomography (CT) scan showed significantly exacerbated infectious lesions with a positive result for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid. An abdomen CT scan indicated the tumor of ascending colon with local wrapped changes. She was diagnosed with ‘Severe novel coronavirus pneumonia’ and ‘Incomplete bowel obstruction: Colon cancer?’. After actively anti-inflammatory and anti-viral therapies, a right colectomy with lymph node dissection was performed on March 11, followed by a pathological examination. The patient successfully recovered from COVID-19 pneumonia and incomplete bowel obstruction after surgery without any postoperative related complications and was discharged on the 9th day after operation. Significant degeneration, necrosis and slough of focal intestinal and colonic mucosal epithelial cells were observed under microscope. No surgeons, nurses or anesthetists in our team were infected with SARS-CoV-2.It is meaningful and imperative to share our experience of protecting health care personnels from SARS-CoV-2 infection and providing references for optimizing treatment of cancer patients, at least for the operative intervention with absolute necessity or surgical emergency, during the outbreak of COVID-19. The rapid spread of the novel coronavirus 2019-nCov, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses on February 12, 2020, has caused an ongoing outbreak of viral pneumonia and pandemic across the world since December 2019, which has been declared as a public health emergency of international concern by the World Health Organization . NotablyA 69-year old Chinese female patient presenting intractable intermittent fever, dry cough, chest tightness and right lower abdomen pain for 2 days was sent to our hospital for further novel coronavirus pneumonia and cancer treatments. On January 28, 2020, the patient developed a 38.4 °C fever, accompanied by fatigue and muscle soreness. This patient denied any medical, trauma or surgical history. Laboratory tests indicated slightly increased neutrophil percentage (74.0%) and decreased lymphocyte percentage 18.6%). A chest computed tomography (CT) scan on February 1 showed a few ground-glass opacities in the lower lobes of both lungs, suggesting suspicion for ‘infectious lesions of both lungs’ , caused by SARS-CoV-2, has attracted not only Chinese but also worldwide concerns [Although the radical right colectomy is a standard surgical procedure for gastrointestinal surgeons, it becomes physically and technically challenging to perform the operation for colon cancer patient with confirmed SARS-CoV-2 infection in the specific period, because of its complexity which includes prolonged operation time and unclear vision, as well as discomfort and inconvenience caused by heavy personal protective equipments (PPEs). More importantly, several reasons give it less clinical priority to perform invasive diagnostic and surgical procedures, such as suddenness of the outbreak, high rate of transmission, vast patient volume in hospitals, and severe shortage of health care personnel , which iA previous study has demonstrated that combining antiviral and anti-inflammatory treatments could simultaneously reduce viral infectivity, viral replication and the aberrant host inflammatory response . TherefoDuring the earlier phase of the COVID-19 outbreak, there were a significant number of health care providers infected with SARS-CoV-2 in many hospitals in Wuhan, and patients in the same room were cross-infected, due to exposure to unknown transmission sources. This dramatically increased the risk of SARS-CoV-2 infection for everyone in the outpatient clinics, especially cancer patients. On retrospective analysis of the patient’s medical course, the underlying cause of the first fever might be tumor-derived, and then were cross-infected with SARS-CoV-2 due to outpatient exposure. The SARS-CoV-2 infection and tumor lesion together caused a synergistic mechanism of activation and further aggravated the patient’s condition, which was partly clarified by the fever eventually reducing to normal after surgery. Pathologic findings revealed that the obvious degeneration, necrosis and slough of focal intestinal and colonic mucosal epithelial cells surrounding the tumor, which was consistent with the findings in a previous autopsy report . Based o


We highlight the strengths of these platforms, including their application to assess both the susceptible cell types and the pathogenesis of SARS-CoV-2. We describe their use to identify drug candidates for further investigation in addition to discussing their limitations in fully recapitulating COVID-19 pathophysiology. Overall, stem cell models are facilitating the understanding of SARS-CoV-2 and prove to be versatile platforms for studying infections.The coronavirus disease 2019 (COVID-19) global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected over 200 countries and territories worldwide and resulted in more than 2.5 million deaths. In a pressing search for treatments and vaccines, research models based on human stem cells are emerging as crucial tools to investigate SARS-CoV-2 infection mechanisms and cellular responses across different tissues. Here, we provide an overview of the variety of human pluripotent stem cell-based platforms adopted in SARS-CoV-2 research, comprising monolayer cultures and organoids, which model the multitude of affected tissues  The majority of infected individuals experience only mild to moderate symptoms that do not require hospitalization  or are ain vitro the diversity of cell types present in human organs. These cell lines also generally carry cancer-associated mutations in genes controlling cell cycle and proliferation  have rapidly emerged as an alternative to animal models as well as to immortalized and cancer human cell lines, since they are human cells that have the ability to self-renew indefinitely and differentiate into cells of the three germ layers. They avoid interspecies differences and can be used to obtain abundant samples of a variety of different cell types. Under precise differentiation conditions, hPSCs, including embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can generate specific cell types in monolayer cultures. In addition, over the last few years numerous differentiation protocols have been developed to generate three-dimensional (3D) cultures, known as organoids, which more faithfully recapitulate human organs Both hPSC-derived monolayer cultures and organoids have already been used to investigate host-virus interactions in different human cell types and tissues, including modelling respiratory infections, such as influenza, enteric infections, such as those due to norovirus and rotavirus, hepatic infections, such as hepatitis B and hepatitis C, infections of components of the immune systems, as in HIV and dengue virus studies, and both prenatal and postnatal brain infections, including those caused by Zika virus and herpes simplex virus 1. In the case of Zika virus, they have also been employed to identify antiviral drug candidates . Several2in vitro have usually been compared to those in primary human tissues, confirming hPSC-derived cells and organoids as reliable models. Indeed, hPSC-derived cells expressing ACE2 and TMPRSS2 or other putative entry receptors and priming proteases become infected with SARS-CoV-2. A common pattern that has been observed across models of different tissues is the increased expression of genes involved in the innate immune response, as chemokines, interleukins and other cytokines upon SARS-CoV-2 infection  and intestinal (PSC-HIOs) organoids derived from hPSCs have been used to investigate the cell type-specific susceptibility to SARS-CoV-2 infection in the gastrointestinal tract . Transcrll types . Colonic-2 virus , in agre-2 virus . Differe-2 virus . Increas-2 virus .2.5As hepatic dysfunction has also been observed in COVID-19 patients , hPSCs-d2.6in vitro or transplanted in mice  organoids . SARS-Crganoids . Upon SArganoids . Also thrganoids , while organoids . These crganoids . Consensrganoids . After Srganoids , that mirganoids . Increasrganoids  as well rganoids , startinrganoids , dysregurganoids , and trarganoids .2.8SARS-CoV-2 tropism and mechanisms of infection have also been investigated using hPSC-derived whole-eye SEAM organoids, comprising cells of the cornea, iris, ciliary margin, lens, retina and retinal pigment epithelium . ACE2 wa3in silico or in vitro screenings of drugs that are commercially available, currently in clinical trials for other pathologies, or already characterized in preclinical studies, have already begun  and alveolar (hALOs) organoids, observing similar results  as a promising target. Treatment of hPSC-CMs with BRD inhibitor INCB054329 at the same time of exposure to SARS-CoV-2 did not reduce viral replication or viral load while pre-treatment with INCB054329 before infection significantly reduced the viral load, decreased the number of infected cells and avoided sarcomere disorganisation, suggesting that BRD inhibitors are promising candidates for further investigation   and vasctigation . Other stigation  or targetigation . To identigation . To findCOVID-19 . Using aCOVID-19 . hrsACE2COVID-19 . TreatmeCOVID-19 .. In agrCOVID-19 . The obsCOVID-19 . Neverth3.3The efficacy of hrsACE2 in reducing SARS-CoV-2 infection has also been validated in kidney organoids . The obs3.4in vitro also in hPSC-based models of gastrointestinal tract and identify EK1 as a new promising drug for further evaluation.As the gastrointestinal tract is another target of SARS-CoV-2 infection, our group tested in hPSC-COs the antiviral efficacy of the three drugs identified as hit compounds in the hAWOs screening described previously . Imatini3.5in silico studies in vitro As more studies adopt and advance hPSC-based platforms to investigate COVID-19 pathophysiology, they will facilitate a better understanding of infection mechanisms and expedite the identification of candidate treatments, complementing the findings obtained from primary human tissues and animal models.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shuibing Chen reports financial support was provided by National Institute of Diabetes and Digestive and Kidney. Shuibing Chen reports financial support was provided by Bill and Melinda Gates Foundation.


An 84-years-old woman with hypertension and dyslipidemia was admitted in the emergency room with acute abdominal pain; patient complained of chest pain three weeks before hospital admission. On physical examination, she was hypotensive, tachycardic (with arrhythmic pulse), tachypneic and with diffuse abdominal pain. Electrocardiogram showed atrial fibrillation with rapid ventricular response, complete left bundle branch block and inferior Q waves. Abdominal computed tomography (CT) revealed a thrombus in the superior mesenteric artery -2 showed1 Occurrence of late myocardial infarction should raise suspicion and clinical signs may be atypical.2Myocardial rupture demands a prompt diagnosis.This case illustrates an interesting entity - pseudoaneurysm, with left-to-right shunt and contained myocardial rupture, which extended to the right ventricle, leading to a dismal prognosis.


The treatment of advanced and metastatic non-small cell lung cancer (NSCLC) has changed dramatically in recent years due to advanced molecular diagnostics and the recognition of targetable oncogenic driver alterations. This has led to the development of very effective new targeted agents, and thus to a relevant progress in the treatment of oncogene-addicted NSCLC. While the treatment of EGFR-mutated and ALK-rearranged NSCLC is well-established, new targeted therapy options have emerged for other oncogenic alterations. In this comprehensive review article, we discuss the major molecular alterations in NSCLC and the corresponding therapeutic options.Due to groundbreaking developments and continuous progress, the treatment of advanced and metastatic non-small cell lung cancer (NSCLC) has become an exciting, but increasingly challenging task. This applies, in particular, to the subgroup of NSCLC with oncogenic driver alterations. While the treatment of epidermal growth factor receptor (EGFR)-mutated and anaplastic lymphoma kinase (ALK)-rearranged NSCLC with various tyrosine kinase inhibitors (TKIs) is well-established, new targets have been identified in the last few years and new TKIs introduced in clinical practice. Even for KRAS mutations, considered for a long time as an “un-targetable” alteration, promising new drugs are emerging. The detection and in-depth molecular analysis of resistance mechanisms has further fueled the development of new therapeutic strategies. The objective of this review is to give a comprehensive overview on the current landscape of targetable oncogenic alterations in NSCLC. Lung cancer is by far the leading cause of cancer-related mortality in both male and female worldwide, accounting for nearly 20% of all cancer deaths in Europe . Most reGenerally, oncogenic driver alterations are mutually exclusive, which means that the presence of one oncogenic driver event excludes the presence of another active oncogenic driver. This concept is based on the model that a single, early genomic driver event leads to a state of oncogene addiction, which drives tumorigenesis and tumor progression. Therefore, the respective proteins are ideal targets for anticancer drugs.Treatment outcome with targeted therapies is superior to conventional chemotherapy. Randomized phase III trials in different settings have shown significantly improved progression-free survival (PFS) and overall survival (OS).Generally, TKIs, selected by a predictive targetable oncogenic driver alteration, yield a high objective response rate (ORR). The magnitude varies, but usually the ORR ranges between 50% and 90% as will be described in the following chapters. Responses to TKIs occur timely after onset of therapy, usually within 4–6 weeks. Emergence of secondary resistance to targeted therapy is inevitable and usually occurs within 12–24 months. Molecular re-analysis at time of progression for characterization of acquired resistance mechanisms has led to new therapeutic options for various molecular subtypes.Although there are a variety of molecular NSCLC subtypes, oncogene-addicted NSCLC share some typical hallmarks and similarities:The present review provides an overview on targeted therapy options in advanced and metastatic NSCLC with oncogenic driver alterations.With significant progress in treatment options for patients with advanced, inoperable stage NSCLC, predictive biomarkers, including the morphological subtype, the PD-L1 status and the results of targetable oncogenic driver alterations, are a prerequisite for the decision on the best possible therapy as they are directly linked to the choice of a specific treatment ,12. ThouGuidelines recommend that all patients with advanced or metastatic non-squamous cell carcinoma of the lung  should be tested for targetable oncogenic driver alterations ,12,13. RTypically, advanced stage lung cancer patients are diagnosed by small biopsies and cytology specimens and generally provide enough tumor material for predictive testing. Of note, cytology specimens as a source for predictive molecular biomarker testing is important, as up to 40% of all lung cancers are diagnosed by cytology alone. Conventional cytology preparations, due to the lack of formalin fixation, yield high-quality DNA and are very well suited for sequencing analyses . CurrentTo detect the increasing number of treatment relevant molecular targets, frontline simultaneous targeted DNA- and RNA-based next generation sequencing (NGS) is most straightforward as a large panel of relevant genes can be tested for mutations, fusions and amplifications in a timely manner . Total nIHC for ALK and ROS1 is widely used and has been introduced more recently for NTRK fusions and can significantly simplify large-scale screening for these rare gene rearrangements ,23,24. ALK IHC, using 5A4 or D5F3 antibody clones, is an equivalent alternative to FISH, which used to be the gold standard for ALK testing . In NSCLThere is no established antibody for the detection of RET rearrangements by IHC and RET testing is performed by break-apart FISH, RNA-based NGS or reverse-transcriptase PCR . Another recently introduced predictive marker, as mentioned above, are MET exon 14 alterations. Genomic alterations in MET exon 14 and its flanking introns cause MET exon 14 skipping during pre-mRNA splicing with loss of the juxtamembrane domain containing the Y1003 residue that serves as a binding site for the E3 ubiquitin ligase CBL. This results in impaired MET degradation with increased half-life of the active MET receptor and extended downstream MET signaling . There aAnother mechanism of oncogenic MET activation is MET amplification . Using FIn the setting of acquired resistance under TKI treatment, resistance mechanisms develop in a subclonal fashion and might vary between different tumor sites . Liquid In summery simultaneous frontline DNA- and RNA-based NGS for mutation, rearrangement and amplification testing is most straightforward. However, testing algorithms might need to be flexibly adapted and methods combined to adjust to the needs of the available tumor material, the available testing methods, the local economic situation and to access to drugs. Molecular re-analysis at time of progression under TKI-treatment allows to adapt the treatment to the changing molecular profile of the tumor. Liquid biopsy is an evolving, minimally-invasive diagnostic tool, which can detect treatment relevant alterations omitting a re-biopsy in a subset of patients. With the increasing complexity of molecular results, multidisciplinary discussion in molecular tumor boards is recommended to put the findings in the appropriate clinical context and to discuss optimal treatment options.The prevalence of oncogenic driver alterations depends on clinical variables  and tumor-associated factors such as histology and stage ,46.In non-squamous histology, the most frequently detected oncogenic driver alterations are KRAS mutations, followed by EGFR mutations, ALK gene rearrangements, BRAF mutations, MET alterations including MET exon 14 skipping mutations and MET amplification, HER2 alterations, RET, and ROS1 gene arrangements and NTRK 1/2/3 gene fusions. While KRAS mutations are the most prevalent oncogenic alterations in metastatic AC (up to 30%), the incidence of EGFR mutations is highly dependent on the tested population 12–47%) 48]. BothIn patients with SCC, the frequency of sensitizing EGFR mutations, ALK, ROS1, and RET rearrangements, MET alterations and BRAF V600 mutations is low and molecular testing not routinely recommended ,52,53. TMolecular oncogenic driver aberrations, their frequency and targeted therapies (in the order discussed below) are summarized in A selection of pivotal clinical trials of targeted agents in patients with oncogenic driver alterations is presented in A graphic overview on molecular oncogenic driver aberrations and targeted therapies is provided in KRAS  oncoprotein is a GTPase and an essential mediator of intracellular signaling pathways, including the RAF-MEK-ERK (MAPK) pathway, involved in cell growth and survival ,56. KRASAlthough various attempts inhibiting KRAS have been made, there is no established therapy for this large patient subpopulation. So far, the most promising approach was the combination of MEK-inhibitors with chemotherapy ,65,66. HVery recently, encouraging signs of efficacy have been observed for two newly developed specific KRAS G12C inhibitors. In the proximity of the mutated cysteine of the KRAS G12C mutation (glycine-to-cysteine mutation at position 12) there is a pocket (P2) that is present in the inactive GDP-bound conformation of KRAS . BindingThe first KRAS G12C inhibitor that reached clinical testing in humans is sotorasib (AMG 510) . Early cThe second KRAS G12C inhibitor that is currently studied in a phase I/II trial (NCT03785249) is adagrasib (MRTX849). The phase I/II KRYSTAL-1 trial included patients with advanced, pretreated solid tumors harboring a KRAS G12C mutation. Overall, 79 patients with KRAS G12C mutant NSCLC have been treated with adagrasib 600 mg bid. Response data are available for 51 patients so far. ORR was 45% and disease control rate (DCR) was 96% . After aNumerous clinical trials with new KRAS inhibitors are currently ongoing. In these trials KRAS G12C inhibitors are compared to conventional chemotherapy or investigated in combination with immune checkpoint inhibitors or other targeted agents.Epidermal growth factor receptor (EGFR) belongs to a family of four receptors  and is the best-known and established oncogenic target in advanced and metastatic NSCLC ,7. The mGefitinib is a first-generation inhibitor of EGFR . After pThe landmark Iressa Pan-Asia Study (IPASS) enrolled treatment-naïve Asian patients with stage IIIB or IV lung adenocarcinoma who had never smoked or were light smokers . PresencThe WJTOG 3405 study enrolled only patients with EGFR mutations and randomized participants to gefitinib or cisplatin plus docetaxel . GefitinFinally, the NEJ002 trial also exclusively included patients with EGFR mutations . ChemothErlotinib is another first-generation inhibitor of EGFR. Erlotinib has been approved for the treatment of metastatic NSCLC even before the discovery and characterization of the EGFR mutations in 2004. The approval based on the positive results of the phase III BR.21 study . In a thThe identification of the EGFR mutation as a predictive biomarker for EGFR-TKIs led to the initiation of three large phase III studies comparing erlotinib to chemotherapy. All trials demonstrated superiority of erlotinib to chemotherapy in patients with EGFR mutation-positive NSCLC ,87,88.The Chinese OPTIMAL study was one of the first trials that compared upfront erlotinib with carboplatin plus gemcitabine in patients with advanced EGFR-mutated NSCLC . Both PFAfatinib is a second-generation TKI that irreversibly inhibits EGFR, ErbB2 and ErbB4 . AfatiniDacomitinib is a second-generation TKI that irreversibly inhibits ErbB/HER receptors including EGFR, HER 1, HER2 and HER4 . The ranAbout 50–60% of patients treated with first-generation TKIs develop a T790M resistance mutation in exon 20 ,107. ThuDifferent other third-generation EGFR inhibitors have been developed to target both standard EGFR-sensitizing and T790M resistance mutations, including rociletinib , abivertAn interesting therapeutic strategy is the concept of combining EGFR-TKIs with other agents with the aim to achieve better and more durable responses in EGFR-mutated patients. The randomized phase II CALGB 30406 trial has been one of the first studies to assess the addition of chemotherapy to erlotinib in EGFR-mutated patients . While eOngoing randomized clinical trials are investigating the addition of chemotherapy (NCT04035486), bevacizumab (NCT02971501) and ramucirumab (NCT03909334) to the third-generation TKI osimertinib.Interesting results from the CHRYSALIS study (NCT02609776) investigating a combination treatment with amivantamab, a bispecific antibody against EGFR and MET, and the third-generation EGFR-TKI lazertinib, have been presented at the 2020 virtual ESMO congress . This muEGFR exon 20 insertions mutations occur in 4–10% of all patients with EGFR mutations. Clinical activity of irreversible EGFR-TKIs in patients with NSCLC harboring EGFR and HER2 exon 20 mutations has been assessed, yielding however disappointing results. The results may be explained due to pharmacokinetics of second-generation EGFR-TKIs as dose-limiting toxicity impeded to attain the inhibitory plasma concentrations of exon 20 insertions. . PatientAmivantamab has proven active in the phase I CHRYSTALIS trial, which included pretreated and treatment-naïve patients with advanced NSCLC harboring EGFR exon 20 insertions . ReductiRecently, data from early phase trial with molecules designed to selectively target EGFR and EGFR2 (HER2) exon 20 insertion mutations have been presented. Mobocertinib (TAK-788), a highly specific TKI, showed anti-tumor activity (ORR 43%) in patients with EGFR exon 20 insertion mutations at the recommended phase II dose (160 mg once daily) . MobocerAnaplastic lymphoma kinase (ALK) is a transmembrane tyrosine kinase receptor that is expressed in neural tissue, the small intestine, and the testes and plays a crucial role in the development of the central nervous system . The ALKp = 0.0978) [Crizotinib was the first targeted drug that showed clinical activity in patients with ALK-positive NSCLC with response rates similar to what has been seen in EGFR mutant NSCLC with EGFR-TKIs . The PRO 0.0978) . Although crizotinib yields high response rates, durable responses are rare and most patients eventually relapse. This led to the development of more specific ALK inhibitors that were able to overcome crizotinib-resistance.Ceritinib is an inhibitor of ALK and ROS1 and demonstrated activity in patients with ALK-rearranged NSCLC who had progressed on crizotinib. In the phase II ASCEND-2 trial, patients who have been previously treated with at least one platinum-based chemotherapy and progressed on crizotinib achieved an ORR of 38.6% . The durAlectinib is a highly selective ALK inhibitor ,145 and Brigatinib is an ALK inhibitor that targets ALK mutations, ROS1 rearrangements, and has preclinical activity against EGFR ,152,153.Lorlatinib is a potent third-generation inhibitor of ALK and ROS1 tyrosine kinases . In phasEnsartinib, a new-generation ALK inhibitor, was investigated in 160 patients with ALK-positive metastatic NSCLC who had progressed on crizotinib therapy [Interim results from the phase III eXalt3 study have been presented at the 2020 IASLC’s World Conference on Lung Cancer Virtual Symposium . The triEntrectinib is an inhibitor of ALK, ROS1 and pan-TRK with activity in ALK-positive NSCLC in early phase studies . Entrectv-Raf murine sarcoma viral oncogene homolog B (BRAF) is a serine/threonine kinase that is part of the MAP/ERK signaling pathway. Activating point mutations in BRAF result in unregulated signaling via the MAP/ERK pathway. BRAF mutations have initially been described in malignant melanoma with 40–60% of tumors harboring an activating BRAF V600E mutation . SubsequThe presence of the BRAF V600E mutation is associated with response to BRAF as well as combined BRAF and MEK inhibitors. BRAF inhibitors have successfully been investigated in BRAF-mutant malignant melanoma . In 2015The efficacy of combined treatment with the BRAF inhibitor dabrafenib and the MEK 1/2 inhibitor trametinib in patients with metastatic BRAF V600E-mutated NSCLC has been demonstrated in an open-label phase II trial (NCT01336634) including previously pre-treated (cohort B) and untreated patients (cohort C). Thirty-six of 57 pre-treated patients (63%) receiving the combination of dabrafenib/trametinib achieved an investigator-assessed objective response . In an uNo data exists to support the use of BRAF/MEK inhibitors for non-V600 mutations and chemotherapy or immunotherapy remain the preferred treatment options. Outcome and response rate to Vemurafenib-monotherapy in patients with non-V600 BRAF mutated NSCLC has been disappointing . Human epidermal growth factor receptor 2 (HER2), also known as ErbB2, is a member of the ErbB receptor tyrosine kinase family. There is no known HER2 ligand and activation is induced by homo- or heterodimerization ,183. ActIn the retrospective European EUHER2 cohort study, the ORR of the pooled TKIs  was 7.4%, while the response rate with trastuzumab-chemotherapy combinations or ado-trastuzumab emtansine was 50.9% . Other pAdo-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) composed of trastuzumab linked to DM1, a cytotoxic microtubule-inhibitory agent. T-DM1 proved effective in a single-center phase II basket trial with HER2-mutated adenocarcinoma, demonstrating an investigator-assessed ORR of 44% and median PFS of 5 months . ResponsAt the 2020 American Society of Clinical Oncology (ASCO) congress, results from the DESTINY-Lung 01 trial, a multicenter, phase II study, investigating the novel ADC trastuzumab deruxtecan (T-DXd) in patients with HER2-mutated NSCLC have been presented . T-DXd iDifferent pan-HER2 TKIs have been evaluated in HER2-altered NSCLC. Afatinib, a TKI with activity against ErbB family members, is approved for EGFR-mutated AC, but has not shown meaningful activity in lung cancer patients harboring HER2-mutations . NeratinInteresting results have been recently presented regarding TKIs that selectively target both EGFR and HER2 exon 20 insertion mutations, including mobocertinib, poziotinib, and tarloxotinib. Mobocertinib has been assessed in a phase I/II trial (NCT02716116), including a cohort of patients with HER2 exon 20 mutated NSCLC. The results have not yet been published. Results from the multi-cohort phase II ZENITH20 trial of poziotinib in patients with EGFR or HER2 exon 20 mutations have been presented at the virtual 2020 ESMO congress . The resThe c-ros oncogene 1 (ROS1) encodes a tyrosine kinase receptor from the insulin receptor family. A rearrangement of ROS1 has initially been described in glioblastoma ,206,207.The phase I PROFILE 1001 study investigated crizotinib, a tyrosine kinase inhibitor of ALK, ROS1 and MET, versus platinum-based chemotherapy in patients with ALK-positive NSCLC . An expaCeritinib, an ALK and ROS1 inhibitor, was investigated in a Korean phase II study, where 32 patients with ROS1-rearranged advanced NSCLC were treated . The ORRActivity against ROS1 has also been described for lorlatinib . In 69 RFor entrectinib, a multikinase inhibitor, a pooled analysis from three phase I-II trials  showed an impressive ORR of 77% and median PFS of 19.0 months . An intrRepotrectinib and Taletrectinib are two new ROS1 inhibitors and have been evaluated in early phase studies for ROS1-positive NSCLC.The ongoing TRIDENT-1 study investigates repotrectinib in patients with advanced ROS1-, TRK-, and ALK-positive solid tumors. First safety and efficacy signals in the cohort of patients with ROS1 positive NSCLC were published in 2019 . ORR wasTaletrectinib is an orally available and potent selective small molecule inhibitor of ROS1 and NTRK with activity in NSCLC patients with ROS1 fusion . In a phRearranged during transfection (RET) is a receptor tyrosine kinase and a known oncogene in thyroid cancer, where translocations, as well as activating mutations, have been detected ,223. ThePatients with RET-fusion positive NSCLC show a high response to pemetrexed-based chemotherapy regimens, possibly due to a lower expression of thymidylate synthetase in the tumor tissue ,231.Different non-selective RET inhibitors have been assessed retrospectively in a large international registry of patients with NSCLC harboring RET-fusions . In thisThe safety and antitumor activity of the multikinase RET inhibitor agerafenib (RXDX-105) has been explored in a phase I/Ib trial . ORR in High efficacy has been demonstrated with two new selective RET inhibitors: selpercatinib (LOXO-292) and pralsetinib (BLU-667).Selpercatinib, has been investigated in the phase I/II Libretto-001 study, with a cohort of 531 patients with RET fusion-positive NSCLC ,242. AmoPralsetinib (BLU-667) is investigated in the multi-cohort phase I/II ARROW trial (NCT03037385) in patients with RET gene fusion-positive tumors including NSCLC patients. The first results were presented at the 2019 ASCO annual meeting . The regMET is a proto-oncogene encoding a receptor tyrosine kinase (c-MET), which undergoes homodimerization by binding its ligand, hepatocyte growth factor (HGF). Homodimerization and autophosphorylation of c-MET leads to the activation of various intracellular signaling pathways including RAS-RAF-MAPK, JAK-STAT and PI3K-AKT-mTOR . Gain-ofFirst therapeutic approaches for MET-altered NSCLC included combination treatments of MET inhibitor tivantinib with erlotinib  and monoCrizotinib, a non-selective MET inhibitor, has demonstrated clinical efficacy both for MET amplifications and MET exon 14 mutations. In the phase I/II PROFILE 1001 trial, 69 patients with tumors harboring MET exon 14 alterations were included . Most ofResponses to crizotinib have also been observed in patients with MET-amplified tumors ,214,258.Capmatinib, a selective MET inhibitor, has been evaluated in the multi-center, multi-cohort phase II GEOMETRY mono-1 study . GEOMETRIn treatment-naïve patients with NSCLC bearing MET exon 14 mutations (cohort 5b), capmatinib demonstrated an ORR of 68%. Median DOR was 12.6 months and mPFS 12.4 months. Patients with MET exon 14 mutations who had prior treatment (cohort 4) had an ORR of 41%, mDOR of 9.7 months and mPFS of 5.4 months. Results from the expansion cohort of pre-treated patients with MET exon 14 mutations (cohort 6) were in line with the efficacy of capmatinib in cohort 4 (ORR 48%). The intracranial activity in patients with NSCLC harboring MET exon 14 mutations (cohort 4 and 5b) was 54%. Activity of capmatinib was also reported in patients with high-level MET-amplified metastatic NSCLC (gene copy number ≥10), although response rates were lower than in patients with MET exon 14 mutations. In treatment-naïve patients the observed ORR was 40% (cohort 5a), and 29% in pre-treated patients (cohort 1a). Median DOR in these patients was 7.5 and 8.3 months, respectively. Most common treatment-related adverse events across all cohorts were peripheral edema , nausea (45% and 2%), and vomiting (28% and 2%). Laboratory alterations included increased blood creatinine in 24% of patients. Tepotinib is another small molecule MET inhibitor. VISION, a multi-center, multi-cohort phase II trial, assessed tepotinib in patients with locally advanced or metastatic NSCLC with MET exon 14 skipping mutation and MET amplifications . Among 1Savolitinib, another selective MET inhibitor, has demonstrated durable clinical activity in patients with NSCLC who harbor a MET exon 14 skipping mutation . ResultsInteresting early results have been shown for Sym015, a synergistic mixture of two recombinant monoclonal antibodies against non-overlapping epitopes of MET . In the Amivantamab (JNJ-61186372), a bispecific EGFR-MET antibody is tested in the CHRYSALIS phase I study (NCT02609776). This study comprised a dose expansion phase in patients with EGFR- and MET-mutated NSCLC.Telisotuzumab vedotin (ABBV-399) is an ADC targeting c-MET and is currently tested in phase II study (NCT03539536).Neurotrophic tyrosine receptor kinase (NTRK) genes encode tropomyosin receptor kinase (TRK) fusion proteins that act as oncogenic driver in different types of tumors . NumerouLarotrectinib has been assessed in an adult phase I trial, a pediatric phase I/II trial (SCOUT) and an adult/adolescent phase II basket trial (NAVIGATE) . The ORREntrectinib was tested in an adult phase I trial (ALKA-372-001), an adult phase I trial (STARTRK-1), a phase II basket trial (STARTRK-2), and a phase I/II pediatric trial (STARTRK-NG) ,269,270.Currently, second-generation NTRK inhibitors, such as repotrectinib  and selitrectinib (NCT03215511), are investigated in clinical studies.Recent developments in the subgroup of NSCLC with oncogenic driver alterations, highlighted in the present review, have brought significant clinical patient benefit and established an individualized treatment approach. The treatment landscape of oncogenic-addicted NSCLC is becoming increasingly complex. The choice of the optimal treatment strategy and management of TKI-therapy, including adverse events, requires expertise and a multidisciplinary approach. Whilst further progress is expected, the prerequisite for this is an ongoing effort to include patients in clinical trials.


In this review, we discuss treatment strategies in biliary tract cancers . In particular, we will describe advances in cellular therapies and cancer vaccines for biliary tract cancers, followed by our local experience with combining a melanoma-associated antigen (MAGE)-positive cell lysate-based autologous dendritic cell vaccine and anti-angiogenic therapy (bevacizumab) in a case of stage IV gallbladder cancer.Biliary tract cancer (BTC) is a rare, but aggressive, disease that comprises of gallbladder carcinoma, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with heterogeneous molecular profiles. Advanced disease has limited therapeutic options beyond first-line platinum-based chemotherapy. Immunotherapy has emerged as a viable option for many cancers with a similar unmet need. Therefore, we reviewed current understanding of the tumor immune microenvironment and recent advances in cellular immunotherapy and therapeutic cancer vaccines against BTC. We illustrated the efficacy of dendritic cell vaccination in one patient with advanced, chemorefractory, melanoma-associated antigen (MAGE)-positive gallbladder carcinoma, who was given multiple injections of an allogenic MAGE antigen-positive melanoma cell lysate (MCL)-based autologous dendritic cell vaccine combined with sequential anti-angiogenic therapy. This resulted in good radiological and tumor marker response and an overall survival of 3 years from diagnosis. We postulate the potential synergism of adding anti-angiogenic therapy, such as bevacizumab, to immunotherapy in BTC, as a rational scientific principle to positively modulate the tumor microenvironment to augment antitumor immunity. Biliary tract cancer (BTC) is a rare but clinically aggressive cancer that accounts for 3–5% of cancer diagnoses globally. It is a heterogeneous disease comprising gallbladder carcinoma (GBC), intrahepatic cholangiocarcinoma (IHCC) and extrahepatic cholangiocarcinoma (EHCC). With an increased understanding of the differing anatomical, histological and molecular subtypes of biliary tract cancers, novel therapies have emerged in the last two decades. However, chemotherapy remains the standard of care in first-line metastatic biliary tract cancers with modest efficacy. As such, more research efforts are required in the subsequent lines of treatment for patients with advanced stage disease.®) with the established gemcitabine-cisplatin regimen proved non-inferiority of the aforementioned regimen [There is still a paucity of highly efficacious chemotherapy to treat advanced BTC. Following the landmark phase III trial ABC-02, combination gemcitabine and cisplatin found first-line indication in the treatment of advanced BTC, but with a median overall survival (OS) of only slightly under a year . More re regimen  or nab-p regimen  has been regimen  in combin = 43) were identified to have potentially actionable mutations, 33% of which had an objective response [The advent of molecular testing and next-generation sequencing (NGS) have unveiled potential treatment options for chemo-refractory disease. Biliary tract cancers have multiple molecular alterations and significant intra-tumoral heterogeneity, with variations of molecular profile across IHCC, EHCC and GBC. Main oncogenic mutation pathways include that of Wnt-Catenin Beta 1 (WNT-CTNNB1), MYC, ErbB, tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) signaling, and this is part of a complex genomic network that modulates cell cycle regulation, DNA damage, MYC amplification, epigenetic regulation, demethylation, kinase signaling and immune dysregulation . In the response . RecentlThe fibroblast growth factor (FGF) pathway upregulates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase-A (PI3KA) pathways; FGF receptor 2 (FGFR2) gene alterations are particularly involved in the pathogenesis of cholangiocarcinoma (CCA) and harbored by about 9–16% of patients. In the FIGHT-202 trial , pemigatIsocitrate dehydrogenase 1 (IDH1) is a key enzyme involved in the citric acid cycle, and mutations in this enzyme have been linked to oncogenesis. IDH1 mutations are present in 25% and IDH2 mutations in 3% of BTC patients, particularly in intrahepatic cholangiocarcinoma . ClarIDHNeurotrophic tyrosine receptor kinase (NTRK) rearrangements are rare in incidence in BTC (<5%), but could still be a potential drug target. Larotrectinib or Entrectinib are approved for patients with such rearrangements in view of durable response rates of up to 75% in a phase II study . There hBTCs usually do not have high mutational burden; in a report of 239 BTC tumors, where whole exome sequencing (WES) was performed, the median numbers of mutations across the IHCC, EHCC and gallbladder cancer subtypes were 39, 35 and 64, respectively, with only 5 patients showing a deficient mismatch repair (dMMR) or microsatellite instability-high (MSI-H) genotype . The MSIEmerging data suggest that in a biomarker unselected population, there can also be durable responses to combination immunotherapy such as Nivolumab and Ipilimumab . GemcitaThe causative link between inflammation and biliary tract cancer has been well established in various types of BTC—the association of gallbladder carcinoma with cholelithiasis-induced chronic inflammation, EHCC with parasites  and chronic pancreatitis and IHCC with persistent hepatolithiasis and hepatitis viruses ,21,22. POne study suggested that chronic inflammation may lead to biliary tract cancers through aberrant activation-induced cytidine deaminase (AID) expression, resulting in the generation of somatic mutations in key cancer genes including TP53, c-MYC and promotor region of INK4A/p16. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interleukin-6 (IL-6) and other cytokines/chemokines have been known to enhance tumor proliferation, activate epithelial-to-mesenchymal transition (EMT) and promote metastatic potential ,25,26. INotably, cholangiocarcinomas are often found to harbor both inflammatory cells  and a high amount of tumor stroma, including vasculature, extracellular matrix (ECM) and fibroblasts including cancer-associated fibroblasts (CAFs) and mesenchymal stromal cells (MSCs) ,30. Key + T cells within the tumor, while more CD4+ T cells were found in the tumor–liver interface [+ or CD8+ T cells [Apart from an immunosuppressive TME, studies also revealed immune escape mechanisms of certain BTCs in the form of major histocompatibility complex-I (MHC-I) downregulation, which was associated with poorer prognosis in BTC and lower tumor-infiltrating lymphocyte (TIL) infiltration . In the nterface , and an  T cells ,39,40.Though immune checkpoint inhibitors have therapeutically revolutionized the field of oncology, their efficacy in advanced BTC is still under active investigation. In view of the limited effective treatment options, there has been increased research in personalized cell-based therapy and cancer vaccines, either as a stand-alone treatment or in combination with other systemic therapies. Among cell therapy approaches, adoptive T-cell therapy, peptide-derived vaccines and dendritic cell (DC) vaccines have emerged with positive clinical outcomes in case reports and early phase clinical trials.Adoptive T-cell therapy refers to the process where patients’ T lymphocytes are extracted and isolated from tumor or blood. They are then activated and expanded in vitro, some with genetic modification, then re-infused usually following lymphodepletion chemotherapy. Such therapies can exist in the form of autologous TIL infusion, chimeric antigen receptor T-cell (CAR-T) therapy targeting specific surface tumor antigens and genetically-modified (GM) T-cell receptor (TCR) T-cell therapy.p = 0.005). Overall survival reached 31.9 months in patients receiving adjuvant immunotherapy compared with 17.4 months (p = 0.022) in patients who did not receive it [n = 9) and pancreatic adenocarcinoma (n = 2) patients treated with HER2-targeted CAR-T therapy demonstrated disease control in 4/9 BTC patients, which included a partial response (PR) that lasted 4.5 months. The infusion of CAR-T-HER2 cells was safe without significant adverse events, except for one grade 3 (G3) febrile event and another G3 transaminitis [A case report in 2006 first suggested the clinical efficacy of adoptive cell therapy in BTC. A patient with IHCC with lymph node involvement received radical resection and was treated with adjuvant CD3-activated T cells plus tumor lysate- or peptide-pulsed dendritic cells and survived over 3.5 years following treatment . Subsequceive it . A recenaminitis . As immunogenic targets, tumor-associated antigens (TAAs) such as Wilms’ tumor protein 1 (WT1) and mucin protein 1 (MUC1) emerged as potential peptide vaccine options in BTC. WT1 mutations were found in about 80% of BTC, while MUC1 was reported to be overexpressed in 90% of BTC. Both are associated with worse prognosis across various cancers . A dose-Subsequently, Human Leukocyte Antigen (HLA)-matched multiple peptide vaccinations were attempted to increase specific antitumor responses. In one case report, whole exome sequencing, whole transcriptomic sequencing and HLA ligandome analyses were used to identify the immunopeptidome that led to the creation of a personalized multi-peptide vaccine (7 non-mutated tumor-associated peptides) emulsified in adjuvant Montanide. This vaccine was administered as multiple dosing to a 56-year-old patient with advanced IHCC, who had also undergone several surgeries for removal of the primary tumor, locoregional recurrences and lung metastasis. She remained recurrence-free for over 41 months . A phaseInterestingly, a phase II trial reported the combined use of Gemcitabine with Elpamotide, an HLA-A*24:02-restricted epitope peptide of VEGF receptor (VEGFR)-2. The reported response rate was 18.5%, with median survival of 10.1 months, which was longer than that of the historical control, and the 1-year survival rate was 44.4%. Injection site reactions were observed in 64.8% of these patients, suggesting the presence of delayed hypersensitivity caused by sensitization of peptide-reactive T cells. In these patients, median OS was significantly longer (14.8 months) compared to those with no injection site reactions (5.7 months) .+ and CD4+ T-cell responses, vaccination did not induce anti-MUC1 antibody responses. Median OS was 26 months with 33% of patients without disease recurrence after 4 years [Dendritic cells are potent professional APCs and effective therapeutic cancer vaccine vehicles that present tumor antigens. The only therapeutic cancer vaccine that has been approved by the FDA is Sipuleucel-T, an autologous active cellular immunotherapy that consists of activated monocytes as APCs presenting a recombinant fusion protein of prostatic acid phosphatase fused to immunostimulant granulocyte-macrophage colony-stimulating factor (GM-CSF) against metastatic castrate-resistant prostate cancer, demonstrating improvement in overall survival . In one  4 years . These r 4 years .Strategies to improve the clinical efficacy of a therapeutic cancer vaccine or adoptive T-cell therapy in BTC include discovery of personalized neoantigens to tailor greater personalized immunotherapy. Preclinical organoid or cell line models may be able to reveal key mutations and mediators that influence TIL infiltration or response to immunotherapy, and can potentially aid in future cell therapy development . SeparatCarcinogenesis in BTC arises through a complex interaction between extracellular ligands and intracellular signaling pathways—an amalgamation of pro-inflammatory cytokines and growth factors as well as aberrant activation and deregulation that orchestrate disinhibited cell proliferation and genetic/epigenetic alterations. It is therefore important to further understand and hence better modulate the tumor immune microenvironment of the BTC to overcome the resistance mechanisms toward cell therapy. Overcoming the immunosuppressive TME through combination approaches such as with immune checkpoint inhibitors, targeted therapy and chemotherapy may potentially improve future treatment outcomes of cell therapy. Our institution, National Cancer Centre Singapore (NCCS), had previously reported a phase II clinical trial of an allogeneic melanoma cell lysate (MCL)-based autologous DC vaccine in 20 metastatic colorectal cancer patients, whose tumors were biopsy-proven for expression of melanoma-associated antigen (MAGE)-A . This DCOur patient presented in December 2013 with recurrent MSS gallbladder cancer with liver, lung and peritoneal metastases. He was 83 years old, had poor performance status (ECOG 2) and had received multiple lines of prior systemic chemotherapy with radiologically proven progression of disease before he was referred to NCCS. He started treatment with DC+Bev in December 2014 and achieved a dramatic reduction in Carbohydrate Antigen (CA) 19-9 and neutrophil-to-lymphocyte (NLR) ratio  after th+HLADR−) population corresponded with cancer progression and treatment—lower during periods of treatment response and higher during pre-treatment and disease progression. The overall CD4 and CD8 T-terminal effector (TEMRA) cells, on the other hand, decreased as the disease progressed  found the cancer to contain KRAS and ABL1 mutations. PD-L1 staining by immunohistochemistry (IHC) showed weak expression. Immune profiling of the serially collected peripheral blood samples showed a shift during the period of treatment response compared to the pre-treatment baseline. Using the Luminex assay, it was observed that, while the pre-treatment plasma cytokine levels of IL-1a, IL-1b, IL-2, IL-6, IL-10, VEGF and FGF2 were all higher than baseline reference ranges for healthy individuals, they were found to decrease during periods of treatment response compared to pre-treatment and disease progression time points . Peripheogressed . Our patient had a good response during 2 years of combined DC vaccine and anti-angiogenic therapy, and survived 3 years from diagnosis of stage IV GBC with a good quality of life. It is noteworthy that this patient had previously received multiple systemic therapies including bevacizumab prior to consulting us at NCCS. We are encouraged by this finding and would like to postulate the potential synergism between anti-angiogenic therapy and the DC vaccine in selected patients, though this needs to be confirmed through larger clinical trials.The addition of the anti-VEGF-A antibody, bevacizumab, to the DC vaccine therapy aims to harness a potential synergistic effect. The mode of action for bevacizumab has been described, including anti-angiogenesis, remodeling the immunosuppressive TME to favor effector T-cell trafficking, infiltration and optimal killing ,61. HoweWe had previous clinical experience of using thalidomide combinations in advanced gastro-esophageal adenocarcinoma  and hepaThere remains an unmet need for BTC patients who are chemo-refractory or do not harbor specific targetable mutations. Encouraging clinical signals are seen in earlier clinical trials of therapeutic cancer vaccination and adoptive T-cell therapy in these patients, indicating a potential, emerging role for cellular immunotherapy against BTC. We believe that our real-world case of an elderly patient with heavily pre-treated GBC who responded to DC vaccination in combination with sequential anti-angiogenic drugs is an example of the nascent yet possible role of cancer vaccines in BTC. It also illustrates the importance of choosing rational combinations to overcome the immunosuppressive tumor microenvironment and augment an antitumor immune effect. The increasing utilization of personalized, tumor-specific antigens with next-generation sequencing and neoantigen predictive platforms harnessed as targets for immunotherapy is also highly anticipated.


In the midst of the SARS-CoV-2/Covid-19 outbreak the need for research into, and development of, antiviral agents is brought into sharp focus worldwide for scientists, governments and the public alike. This special issue includes primary research into new antiviral agents with activity against a range of clinically and economically important viruses. New antiviral strategies are discussed in six review articles.SARS-CoV-2 is a newly emerged β-Coronavirus with sustained human-to-human transmission via the respiratory tract. Influenza viruses are another group of respiratory viruses, which account for 300–600 K deaths annually and represent an ever-present pandemic threat. Emergence of drug-resistant influenza strains is driving research into new anti-influenza drug candidates with different modes of action. In this issue, two papers report compounds with anti-influenza activity against a range of influenza A viruses that inhibit early steps in influenza replication not targeted by clinically approved anti-influenza drugs ,2. A furDrug resistance is an ongoing issue in the global rollout of combination antiretroviral therapy for human immunodeficiency virus (HIV) prevention and treatment. Tadesse et al. report the prevalence of pretreatment drug resistance among HIV-infected children in the resource-limited setting of Ethiopia . LectinsAntiviral agents are not available against many emerging and neglected viruses. This issue features four research papers reporting new antiviral compound candidates against zika virus ,8, hantaIn addition to study into the zoonotic herpes B virus, this issue also includes two primary research papers investigating antiviral approaches targeting reactivation of latent herpesvirus infection. Anderson et al. demonstrated that the antipsychotic drug cloazapine inhibits epstein-barr virus (EBV) lytic reactivation . In contResearch into antiviral agents against economically important animal viruses is also included in this issue, with three primary papers reporting antiviral agents with inhibitory activity against porcine circovirus type 2 (PCV2)  and porcAs mentioned, this special issue includes reviews into antiviral strategies against HIV, measles and CMV ,11,14,15Finally, I would like to thank all the authors, reviewers and editors, who made this issue possible, both at Viruses and in the wider academic community.


Klebsiella pneumoniae (KPC-KP), a type of carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE), from urine specimens collected on July 17 and July 23 from two residents of a ventilator-capable unit in a skilled nursing facility. CP-CRE are multidrug-resistant organisms typically isolated from persons with a health care exposure  Department of Public Health (MCDPH) identified two isolates of carbapenemase-producing MCDPH recommended that the ventilator-capable unit perform contact screening for KPC-KP colonization by rectal swab and culture. The skilled nursing facility had 192 resident beds, 48 (25%) of which were in the ventilator-capable unit; the average length of stay was 14 days. A case was defined as isolation of KPC-KP with a PFGE pattern indistinguishable from that of the two index patients from any specimen source collected from a resident of the ventilator-capable unit during July–November 2018. Contacts were defined as residents residing for ≥3 days in the same ventilator-capable unit as either of the two index patients. On August 13, among 42 identified contacts, six (14%) declined screening, seven (17%) had been discharged, two (5%) were deceased, and one (2%) had a recent infection with a different carbapenem-resistant organism. Among the remaining 26 (62%) residents who were screened, KPC-KP isolates were detected in five (19%) asymptomatic contacts, three of which had indistinguishable PFGE patterns from those of the two index patients.On September 6, MCDPH and ADHS conducted a site visit to the facility to observe infection control practices with emphasis on the ventilator-capable unit and recommend targeted control measures. Observations included missed opportunities for hand hygiene before and after physical contact with residents and lapses in aseptic technique during routine sterile procedures. MCDPH recommended housing residents with CP-CRE infection in the same ward or in the same room when possible; implementing contact precautions with room restriction for residents with CP-CRE infection who are mechanically ventilated, have tracheostomies, or have uncontained body fluids; requiring staff members to perform hand hygiene with alcohol-based hand sanitizer before and after physical contact with residents; increasing access to alcohol-based hand sanitizer by installing additional dispensers; and offering trainings to staff members for commonly performed sterile procedures.On November 5, contacts were rescreened to determine whether recommended control measures were successful in containing the cluster. Twenty-eight residents, none of whom had had KPC-KP isolates detected previously, were identified using the previous criteria for rescreening; nine (32%) declined and 19 (68%) consented, 10 of whom had been screened previously. All 19 (100%) rescreened cultures were negative for KPC-KP. Both index patients were treated with antibiotics for KPC-KP urinary infections, and neither died. Following this investigation, one patient had multiple urine specimens collected in which a KPC-KP isolate was identified, suggesting urinary colonization.Among 26 screened ventilator-capable unit contacts, the investigation identified three (12%) additional cases of KPC-KP colonization with an isolate that had an indistinguishable PFGE pattern from that of the two index patients, which supported health care–associated transmission. Closer adherence to CDC recommendations that could prevent health care–associated KPC-KP transmission include housing together residents with infection, improving adherence to hand hygiene, using gowns and gloves when interacting with residents who require mechanical ventilation or have tracheostomies, and implementing contact precautions for uncontained body fluids (


ARN) of ARN GABA neurons (GABAARN) is also affected in prenatally androgenized (PNA) PCOS-like NPYARN reporter mice . PCOS-like mice and controls were generated by exposure to di-hydrotestosterone or vehicle (VEH) in late gestation. τGFP-expressing NPYARN neuron fiber appositions with GnRH neurons and gonadal steroid hormone receptor expression in τGFP-expressing NPYARN neurons were assessed using confocal microscopy. Although GnRH neurons received abundant close contacts from τGFP-expressing NPYARN neuron fibers, the number and density of putative inputs was not affected by prenatal androgen excess. NPYARN neurons did not co-express progesterone receptor or estrogen receptor α in either PNA or VEH mice. However, the proportion of NPYARN neurons co-expressing the androgen receptor was significantly elevated in PNA mice. Therefore, NPYARN neurons are not remodeled by prenatal androgen excess like the wider GABAARN population, indicating GABA-to-GnRH neuron circuit remodeling occurs in a presently unidentified non-NPY/AgRP population of GABAARN neurons. NPYARN neurons do, however, show independent changes in the form of elevated androgen sensitivity.Polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility, is associated with altered signaling within the hormone-sensitive neuronal network that regulates gonadotropin-releasing hormone (GnRH) neurons, leading to a pathological increase in GnRH secretion. Circuit remodeling is evident between GABAergic neurons in the arcuate nucleus (ARN) and GnRH neurons in a murine model of PCOS. One-third of ARN GABA neurons co-express neuropeptide Y (NPY), which has a known yet complex role in regulating GnRH neurons and reproductive function. Here, we investigated whether the NPY-expressing subpopulation (NPY Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility, affecting 8% to 10% of women of reproductive age , and 90%ARN) in PNA mice also exhibits a reduction in progesterone receptor expression, indicating reduced sensitivity to this important feedback cue .ARN neuron cell bodies, they did not co-localize with NPYARN neurons. The mean total number of GFP-ir NPY neurons was not different between VEH-treated and PNA-treated mice in the ARN as a whole -ir was scattered around the entire ARN , top. Hi a whole , nor in  a whole . No diff a whole  within t a whole . The co-ARN-to-GnRH neuron circuit. Using transgenic reporter expression specific to NPYARN neurons, we identified extensive NPYARN neuron projections to GnRH neurons, particularly to those in the rPOA and AHA. Confocal analysis of close appositions between NPYARN neurons fibers and GnRH neurons found no differences in the NPYARN-to-GnRH neuron projection in PCOS-like PNA females. These findings suggest that this subset of GABAARN neurons are distinct to those that are remodeled by prenatal androgen excess [ARN neurons were found to have virtually no co-expression with PR and ERα, irrespective of prenatal treatment, we did observe a greater proportion of NPYARN neurons co-expressing AR in PNA-treated mice, suggesting an upregulation of AR in NPYARN neurons in the PCOS-like condition. These findings indicate that although the NPYARN population are sensitive to androgens in adulthood, the NPYARN-to-GnRH anatomical circuit is not obviously remodeled following prenatal androgen excess.The present study assessed the impact of prenatal androgen excess, which models PCOS features, on the NPYn excess , 16, 18.ARN neurons and their projections [To dissect NPY/AgRP neurons and their full projections specifically originating from the ARN, AgRP-Cre mice were crossed with a line promoting Cre-dependant τGFP expression , 43. Thijections . In addijections . This mojections , 20, witjections , 54. Thijections . HoweverARN fibers, while only 5% of GnRH neurons in the MS subpopulation received NPYARN fiber contacts. In contrast, tract-tracing of the whole GABAARN population has found that approximately half of the MS GnRH neurons receive close appositions [ARN subpopulations have different projection patterns with respect to the specific GnRH neurons they innervate. This differential pattern of innervation to the GnRH neurons does not appear to have been previously reported, and supports an ongoing yet unproven hypothesis that GnRH neurons at different regions along their anatomical axis represent functional subpopulations. For example, c-Fos expression suggests that the activity and plasticity associated with LH surge generation is restricted to the rPOA subpopulation [The vast majority of GnRH neurons within the rPOA and AHA subpopulations received close contacts from NPYositions . This supulation , 56 and pulation , 58.ARN neurons was strikingly similar between PNA and VEH groups regardless of GnRH neural subpopulation or whether the soma or dendrite was examined. This analysis was refined further, where the density of contacts was assessed to normalize for variations in soma circumference between GnRH neurons, and by looking at 15-µm sections of the primary dendrite. In this instance, again, there were no differences between the VEH and PNA groups. The proximal GnRH neuron dendrite was the focus of this study, as GABAergic remodeling is restricted to this region [ARN-to-GnRH neuron circuit, despite NPYARN neurons composing a large proportion of this circuit [ARN neurons must be undergoing plastic reorganization as a result of PNA treatment, while NPYARN neurons are a distinct, unchanged population.Putative innervation of GnRH neurons by NPYs region , 16. Hows region . In any  circuit , 31. ThiA receptors [ARN inputs to GnRH neurons promotes LH secretion [1 or Y4 receptors to inhibit or excite GnRH neural firing rate [1R at a far greater affinity than Y4R [ARN neurons inhibits LH secretion and slows LH pulse frequency in castrated animals [ARN subpopulation of GABAARN neurons to GnRH neurons in PNA mice aligns with their hyperactive hypothalamic-pituitary-gonadal axis state.Increased GnRH neuronal activity in PCOS-like PNA mice , 19, 21 eceptors , and selecretion , NPY anding rate ; howeverthan Y4R , 62, so than Y4R , but alsthan Y4R . In ovarthan Y4R . In addi animals . TherefoARN neuron population. Male mice and rats have a larger population of NPYARN neurons compared with females [ARN remodeling in the present study may suggest that this population is protected from androgen-driven plasticity in the developmental window that PNA treatment is applied. Although NPY/AgRP neurons are born on approximately embryonic day 12, significant development in this circuit occurs during the first 3 weeks of the postnatal period [Androgens can modulate the development of the NPY females , 65. Lik females , and pre females . NPY/AgR females , prenatal period .ARN neurons specifically. Immunohistochemistry confirmed reduced PR-expressing neurons within the ARN as reported previously [ARN co-expression with ERα or PR. Kim et al demonstrated a similarly low co-expression of ERα via immunohistochemistry in mice while also demonstrating that estradiol reduces NPY mRNA levels [ARN neurons found here is not surprising given the lack of ERα, and is in line with evidence in the ewe showing that NPY mRNA is not affected by progesterone administration, nor do NPYARN neurons possess PR [ARN neurons that lose PR expression in PNA mice [As PCOS-like PNA mice exhibit impaired steroid hormone negative feedback and reduced PR expression in the ARN , 20, we eviously . Consisteviously , 69, we A levels . Estradissess PR . These rPNA mice  cannot cARN neurons and upregulated by PNA treatment. As circulating testosterone levels are elevated in PNA-treated mice [ARN neurons. It remains to be determined whether elevated AR expression impacts neuroendocrine regulation in the PNA-treated mouse. Chronic DHT exposure from 3 weeks of age induces a range of metabolic effects including increased body mass and greater adiposity, and neuron-specific knockout of AR protects against DHT-induced metabolic and reproductive impairments [ARN neurons to increase orexigenic drive. However, prenatal androgen exposure that leads to postpubertal hyperandrogenism does not increase body weight and results in only very mild metabolic disturbances [ARN neurons restrain LH secretion [ARN activity would be at odds with the increased LH pulse frequency present in PNA-treated mice [ARN neurons and whether these actions play a role in the PCOS-like neuroendocrine impairments associated with prenatal androgen excess.In contrast to ERα and PR, AR was both co-expressed in NPYted mice , and AR ted mice , 73, it airments . Thus, eurbances , 54, sugurbances , and actecretion , elevateted mice . TherefoARN innervation to GnRH neurons appears unaltered, it remains possible that alterations to GnRH neuronal afferents are present; for example, NPYARN neurons project to and regulate kisspeptin neurons in the ARN [ARN neurons appear androgen-sensitive, this raises questions regarding the possible functional significance of this increase. As it stands, there is very little information in the mouse to suggest how androgens regulate NPYARN neurons. Future studies will be needed to determine whether androgens acting via AR in NPYARN neurons have any role in the interference of steroid hormone negative feedback within the PNA-treated mouse.While NPY the ARN . Closer