We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide–VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors  implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased. Subcutaneous administration of KS-133 NPs once every 3 days had stronger anti-tumor effects than daily subcutaneous administration of KS-133. Furthermore, KS-133 NPs significantly enhanced the pharmacological efficacy of an immune checkpoint-inhibiting anti-PD-1 antibody. A pharmacokinetic study suggested that the enhancement of anti-tumor activity was associated with improvement of the pharmacokinetic profile of KS-133 upon nanoformulation. Our data have revealed that specific blockade of VIPR2 by KS-133 has therapeutic potential for cancer both alone and in combination with immune checkpoint inhibitors. Vasoactive intestinal peptide receptor 2 (VIPR2) is a class-B G protein-coupler receptor that participates in various physiological function by interacting with its ligand vasoactive intestinal peptide (VIP). VIP–VIPR2 signaling has attracted attention as a drug target in the fields of central nervous system diseases, oncology, and immunity. In 2021, we generated 13-mer bicyclic peptide KS-133 as a potent and selective antagonist to VIPR2 . In addiIn addition to the aforementioned study, we reported that VIPR2-mediated signaling contributes to the polarity of tumor-associated macrophages (TAMs) . Activatin vivo. Generally, peptides are rapidly degraded by proteases, and even if they are resistant to degradation, they are prematurely excreted from the body via renal filtration. Thus, frequent dosing is needed, reducing patients’ quality of life. KS-133 is stable in plasma for at least 24 h [in vivo. A versatile method for improving the pharmacokinetics of peptides is conjugation with polyethylene glycol [in vitro and the potent anti-tumor effects of KS-133 nanoparticles (NPs) when administered alone or combined with an anti-PD-1 antibody.A hurdle in the development of peptides as therapeutic drugs is their low stability e glycol , 16, bute glycol , 18. Thee glycol , 20. In 6) were grown in T75 flask with 20 mL of RPMI medium containing 10% FBS for 3 days. The culture medium (CT26-CM) was collected and centrifuged at 400 g for 5 min. RAW264.7 cells (2.4 × 105) were incubated in 24-well plate in the presence or absence of KS-133  with 20% CT26-CM/DMEM for 3 days. During the incubation, medium containing KS-133 was replaced every day. mRNA extraction was performed using ISOGEN  on day 3 after CT26-CM incubation, and cDNA was prepared. The gene expression of M1 markers , M2 markers , and β-actin was analyzed by real-time polymerase chain reaction (PCR) using a 7300 Fast Real-Time PCR System  and Light Cycler Fast Start DNA Master kit (Applied Biosystems). The relative mRNA expression was determined using the 2−ΔΔCt method. ΔΔCt was calculated using control group data.The detailed method and DNA primer sequences were described previously . RAW264.KS-133 and KS-133(P3S) (a substitution of proline at position three of KS-133 with serine) were synthesized at SCRUM Inc.  using Fmoc-based solid-phase peptide synthesis as previously reported . First, Negative staining was used to observe the morphology and structure of NPs using a high-resolution transmission electron microscope  at an acceleration voltage of 100 kV. The particle size of NPs was measured in a disposable cuvette by dynamic light scattering using Zetasizer Nano ZSP .Guide for the Care and Use of Laboratory Animals [5) were subcutaneously injected into the left groin region of BALB/cCrSlc female mice  under isoflurane anesthesia. KS-133 (30 mg/mL)/DMSO was diluted in D-PBS   , and KS-133 (20 mg/mL)/DMSO (10%)/CrEL (10%) solution was diluted in D-PBS  and injected at 100 μL (30 μg)/mouse subcutaneously every day or every 3 days to tumor-bearing mice for 3 weeks. For anti-PD-1 monotherapy or combination treatment, GoInVivo Purified anti-mouse PD-1 (9.98 mg/mL)  was diluted in D-PBS (antibody concentration = 1.0 mg/mL) and injected at 100 μL (200 μg)/mouse intraperitoneally three times in a week. The tumor growth rate was recorded on days 1, 7, 14, and 21 by measuring the major and minor axes with a digital caliper. Measurements were transformed into tumor volumes using the following formula: tumor volume (mm3)  =  major axis × minor axis2 × 0.5. Thereafter, animals were killed by overdose of pentobarbital sodium and tumor tissues were collected. According to the regulations established by the Animal Care and Use Committee of Kagawa University, it is recommended that the experiment be stopped from an animal welfare perspective when the tumor size exceeds 10% of the body weight, in which case the animal is euthanised and deemed dead. If the tumour was large and the animal appeared to be suffering, Ibuprofen (30 mg/kg) were used.All experimental procedures that involved animals and their care were conducted in compliance with the  Animals  and ARRI Animals , 24. TheThese animal experiments were approved by the Animal Care and Use Committee of Osaka University  and conducted at Osaka University in accordance with the aforementioned guidelines. LC–MS/MS data were obtained using an LCMS-8060NX MS system  connected to a Nexera X3 UHPLC system (Shimadzu) equipped with a Peptide BEH C18 column . Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The elution profile of mobile phase B was 5% isocratic between 0 to 1 min, linear gradient from 5% to 50% between 1 to 1.5 min, and increase from 50% to 75% between 1.5 to 3 min. The flow rate was 0.2 mL/min. For pharmacokinetic studies, free or nanoformulated KS-133 was administered intravenously or subcutaneously to male ICR mice at 1 nmol/g body weight. Serial blood samples were collected from the tail vein using heparinized capillary tubes at the indicated times after administration. The plasma (5 μL) obtained from each blood sample was mixed with 10 μL of 0.1% formic acid in 50% (v/v) acetonitrile/water, and subsequently, 50 μL of acetonitrile was added. After centrifugation, aliquots (50 μL) of the supernatant were mixed with equal volumes of 0.2% formic acid in water and then injected into the LC–MS/MS system. Pharmacokinetic parameters were obtained from the plasma concentration–time data by non-compartmental analysis using Phoenix WinNonlin Software .p < 0.05 was considered statistically significant.Multiple-group comparisons were made using one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. All statistical analyses were performed using GraphPad Prism 6 . TNFα, iNOS, and CXCL10 as M1-type macrophage markers and Mrc-1, IL-1Rn, and CCL-22 as M2-type macrophage markers. As presented in iNOS and CXCL10 (p < 0.05). Conversely, KS-133 significantly decreased the mRNA expression of Mrc-1 (p < 0.05). Therefore, VIPR2 inhibition by KS-133 changes the macrophage polarity toward M1.It has been reported that when RAW264.7 cells are incubated in the presence of the culture supernatant of CT26 cells, M1-type macrophage marker mRNA expression is decreased, and that of M2-type macrophages is increased [p < 0.001). Setting the tumor volume of the control group as 100%, the tumor volumes of the KS-133 monotherapy, anti-PD-1 antibody monotherapy, and KS-133/anti-PD-1 antibody combination groups were 66%, 50%, and 32%, respectively. The results in Figs It has been reported that daily subcutaneous administration of VIPhyb, an antagonist of both VIPR1 and VIPR2, induces anti-tumor effects against CT26 cells implanted to severe combined immunodeficient (SCID) mice and enhances the pharmacological effects of an anti-PD-1 antibody . We evalFirst, we sought to improve the water solubility of KS-133 by amino acid substitution. Using reported VIPR2/KS-133 complex model information , we replThe mechanism of the enhanced water solubility was considered to be the incorporation of the peptide into NPs. Transmission electron microscopy  and partin vivo. As presented in We evaluated whether KS-133 NPs exhibit anti-tumor effects in vivo evaluation was performed  from plasma were 0.68 and 1.95 h, respectively. The longer t1/2 following subcutaneous administration was attributed to the absorption process from subcutaneous tissue to blood. t1/2 of KS-133 NPs  was 2.24 h. There was no significant difference in the maximum drug concentration (Cmax), time to reach maximum drug concentration (tmax), t1/2, area under the plasma concentration-time curve from time 0 to t (AUC0-t), and time 0 to infinity using predicted plasma concentration at time t  of KS-133 with or without nanoformulation. However, the mean residence time  of KS-133 NPs was 3.35 h, versus 2.90 h for KS-133 (p < 0.05). Thus, nanoformulation slightly improved the pharmacokinetic profiles of KS-133, which may have contributed to the improved pharmacological efficacy of the nanoformulation  effect of NPs [Another interesting result was the relationship between the stability and anti-cancer activity of NPs containing a VIPR2 antagonist. In general, nanoformulation technology has been developed as a DDS for nucleic acid medicines targeting intracellular genes , 20. Thut of NPs , 20. In in vitro. Our data have also revealed that KS-133 with or without nanoformulation has anti-tumor efficacy both alone and in combination with an immune checkpoint-inhibiting anti-PD-1 antibody. In the future, KS-133 (including the nanoformulated form) is expected to be used in combination with immune checkpoint inhibitors and other drugs for treatment-resistant cancers.In conclusion, we have demonstrated that KS-133 changes the polarity of macrophages to M1 S1 Table(PDF)Click here for additional data file.


In low- and middle-income countries (LMICs), hospitals can rarely utilize their own antimicrobial resistance (AMR) data in a timely manner.To evaluate the utility of local AMR data generated by an automated tool in the real-world setting.From 16 December 2022 to 10 January 2023, on behalf of the Health Administration Division, Ministry of Public Health (MoPH) Thailand, we trained 26 public tertiary-care and secondary-care hospitals to utilize the AutoMated tool for Antimicrobial resistance Surveillance System (AMASS) with their own microbiology and hospital admission data files via two online meetings, one face-to-face meeting and online support. All meetings were recorded on video, and feedback was analysed.Escherichia coli (CREC) was 129 (range 0–1204), by carbapenem-resistant Klebsiella pneumoniae (CRKP) was 1306 (range 0–5432) and by carbapenem-resistant Acinetobacter baumannii (CRAB) was 4472 (range 1460–11 968) per 100 000 patients tested for hospital-origin BSI. The median number of all-cause in-hospital deaths with hospital-origin AMR BSI caused by CREC was 1 (range 0–18), by CRKP was 10 (range 0–77) and by CRAB was 56 (range 7–148). Participating hospitals found that the data obtained could be used to support their antimicrobial stewardship and infection prevention control programmes.Twenty-five hospitals successfully generated and shared the AMR reports with the MoPH by 28 February 2023. In 2022, the median frequency of hospital-origin bloodstream infections (BSIs) caused by carbapenem-resistant Local and timely AMR data are crucial for local and national actions. MoPH Thailand is inviting all 127 public tertiary-care and secondary-care hospitals to utilize the AMASS. Using any appropriate analytical software or tools, all hospitals in LMICs that have electronic data records should analyse and utilize their data for immediate actions. In March 2023, only the cumulative antibiogram from each participating hospital up to 2020 is available online.5 The cumulative antibiograms are not stratified by infection origin, do not include other parameters  and could not be easily compared between hospitals or areas.In low- and middle-income countries (LMICs), there is some national coordination for an AMR surveillance system; however, having a system that is able to generate local and reliable information on AMR and feedback to local hospitals in a timely manner is still a challenge. For example, in Thailand, 92 surveillance sites participated in the National Antimicrobial Resistance Surveillance Thailand (NARST) and 10 surveillance sites provided data to Global Antimicrobial Resistance Surveillance System (GLASS) in 2020.6 The automatically generated reports stratify by infection origin into community origin and hospital origin based on the recommendations of WHO’s GLASS,7 and provided additional reporting on mortality (%) and total number of deaths with AMR and non-AMR bloodstream infections (BSIs). We recently reported the difference of frequency of AMR BSI between tertiary-care hospitals (TCHs) and secondary-care hospitals (SCHs) using the AMASS v2.0 and retrospective data from 2012 to 2015.8The AutoMated tool for Antimicrobial resistance Surveillance System (AMASS), an offline application to generate standardized AMR surveillance reports from routinely available microbiology and hospital data files, was developed and independently tested in seven hospitals in seven countries.9 In addition, the data of patients with AMR infection can be used as measures/indicators to monitor and guide the antimicrobial stewardship (AMS)10 and IPC programmes.11 Therefore, we consider that the availability and timeliness of local AMR data is one of the most important aspects for local hospitals, national authorities and policymakers. Here, we aim to evaluate the utility of local AMR data generated by the AMASS in the real-world setting in Thailand.The national action plan on AMR in Thailand aims to reduce AMR morbidity by 50%, and the analysis of baseline data from target hospitals is included in the plan.2. Health regions in Thailand were divided into 12 groups of provinces plus Bangkok, totalling 13 regions, using the concept of decentralization.12 Health Administration Division, Ministry of Public Health (MoPH) Thailand, supervised 127 public TCHs and SCHs in health regions 1 to 12 countrywide. These included 35 advanced-level referral hospitals , 55 standard-level referral hospitals  and 37 mid-level referral hospitals .13 Based on the WHO definitions, class A hospitals are generally TCHs and referred to as regional hospitals,14 and classes S and M1 are generally SCHs and referred to as provincial or large district hospitals. All class A and S hospitals, and most of class M1 hospitals, are equipped with a microbiology laboratory capable of performing bacterial culture using standard methodologies for bacterial identification and susceptibility testing provided by the Bureau of Laboratory Quality and Standards, Department of Medical Sciences, MoPH, Thailand.In 2022, Thailand had a population of 66.1 million, consisted of 77 provinces, and covered 513 120 kmFrom 16 December 2022 to 10 January 2023, on behalf of the Health Administration Division, MoPH, we invited and trained 26 TCHs and SCHs to utilize the AMASS with their own microbiology and hospital admission data files via two online meetings, one face-to-face meeting and online support. These 26 hospitals were invited because they were highly active in previous activities with the MoPH; 1 to 4 hospitals were selected from each health region, and the MoPH aimed to have them train other hospitals to utilize the AMASS in the next phase.6 and (iii) informing participants about data files that they needed to export from their own hospital information system (HIS) and laboratory information system (LIS) to be used in the next session.The first online meeting focused on (i) informing participants about the AMASS; (ii) teaching participants to be able to download, unzip and successfully run the AMASS with the example datasets provided in the AMASS;The second online meeting focused on supervising hospitals to run the AMASS with their own hospital datafiles; including how to (i) complete the data dictionary files of the AMASS; (ii) verify the log files to understand whether the AMASS automatically imported and analysed the data accurately or not; and (iii) read the AMR surveillance reports automatically generated by the AMASS. We emphasized that hospitals should not transfer raw patient-level data files to the study team.Escherichia coli (CREC), carbapenem-resistant Klebsiella pneumoniae (CRKP) and carbapenem-resistant Acinetobacter baumannii (CRAB) with other participating hospitals; (iii) and discussion on how to utilize the local and timely AMR reports generated by the AMASS for local actions.The face-to-face meeting focused on (i) validation of the generated AMR reports; (ii) comparison of multiple AMR parameters  of each microbiology laboratory and HIS to participate. For the face-to-face meeting, we additionally requested representatives from AMS and IPC teams to participate.In the second online and face-to-face meetings, we asked participants whether the data in their AMR reports generated by the AMASS were proportional to their workload  and their AMR situation  they observed in their own hospitals. We also asked whether they found the AMASS and the AMR reports useful or not.15In the face-to-face meeting in January 2023, we asked each participating hospital to generate the AMR surveillance report using their own hospital admission and microbiology laboratory data files of the year 2022 and the AMASS v2.0.This was a retrospective study describing the epidemiology of AMR among participating TCHs and SCHs in Thailand, and the utility of local and timely data generated by the AMASS. All meetings were recorded on video, and feedback was analysed by P.K. and K.T.7 Patient hospital number (HN) was automatically used as a record linkage between the microbiology and hospital admission data files. A deduplication process was automatically conducted in which only the first isolate of a species per patient per specimen type per survey period was included.AMASS analysed the data and generated reports based on the recommendation of the WHO GLASS.E. coli (3GCREC) and K. pneumoniae (3GCRKP), CREC, CRKP, carbapenem-resistant Pseudomonas aeruginosa (CRPA) and CRAB, which are in the WHO GLASS priority list of AMR bacteria and are locally important. Only blood culture isolates were included in the analysis of the AMASS v2.0 and in this study.For AMR infections, we analysed the following organisms: MRSA, third-generation cephalosporin-resistant Community-origin  BSIs were defined for patients in the hospital within (longer than) the first two calendar days of admission when the first blood specimen culture positive for a pathogen was taken, with calendar day one equal to the day of admission. Patients were considered at risk for hospital-origin BSI after they stayed in the hospital for more than two calendar days.6 and per 100 000 admissions during the reporting period. The frequency of hospital-origin AMR BSI was calculated as the total number of new patients with hospital-origin AMR BSI per 100 000 patients tested for hospital-origin BSI ,6 per 100 000 admissions and per 100 000 patient-days at risk for hospital-origin BSI during the reporting period. All-cause in-hospital mortality was defined using the discharge summary regularly completed by the attending physicians and reported to the MoPH.The proportion of AMR (%) was calculated as the percentage of patients with new AMR BSI over all patients with new BSIs for the pathogen of interest during the reporting period. The frequency of community-origin AMR BSI was calculated as the total number of new patients with community-origin AMR BSI per 100 000 patients tested for community-origin BSI P values were given to two significant figures, and no longer than four decimal places (e.g. P < 0.0001). Box and dot plots were used to illustrate the distribution of data. We used STATA  for the final statistical analyses and R version 4.0.5 for figures.We compared all continuous variables using the Mann–Whitney test. https://doi.org/10.6084/m9.figshare.22339933.The anonymous AMR surveillance reports generated from participating hospitals are open access and available at Ethical permission for this study was obtained from the Institute for the Committee of the Faculty of Tropical Medicine, Mahidol University (TMEC 23-011). Individual consent was not sought from the patients as this was a retrospective study, and the Ethical and Scientific Review Committees approved the process.Of 26 hospitals invited to the early implementation of the AMASS, all generated and shared their AMR surveillance reports for the year 2022 with the MoPH by 28 February 2023. However, the AMR report from anonymous hospital no. 1 was excluded because the data did not pass validation by the study team. The AMR reports of the other 25 hospitals were validated and included in further analysis  and 10 (42%) were classified as TCHs and SCHs, respectively. Of 10 SCHs, 9 and 1 were classified as class S and M1 by the MoPH Thailand. The median bed number was 860 (range 527–1387) in TCHs and 414 (range 282–535) in SCHs (JAC-AMR Online).In 2022, for community-origin BSI, the median proportion of MRSA was 6% (range 0%–23%), of 3GCREC was 34% (range 18%–49%), of 3GCRKP was 22% (range 10%–46%), of CREC was 1% (range 0%–25%), of CRKP was 7% (range 1%–20%), of CRPA was 14% (range 0%–79%) and of CRAB was 50% (range 13%–100%). The median proportion of AMR was not different between TCHs and SCHs for all pathogens , of 3GCREC was 58% (range 35%–79%), of 3GCRKP was 67% (range 0%–92%), of CREC was 7% (range 0%–42%), of CREKP was 37% (range 0%–72%), of CRPA was 36% (range 0%–100%) and of CRAB was 87% (range 61%–94%). The median proportion of AMR for 3GCRKP, CREC and CRKP was higher in TCHs than in SCHs , by 3GCREC was 1039 (range 333–2525), by 3GCRKP was 260 (range 79–527), by CREC was 50 (range 0–325), by CRKP was 75 (range 19–265), by CRPA was 41 (range 0–1556) and by CRAB was 137 (range 34–294). The median frequency of AMR BSI was not different between TCHs and SCHs for all pathogens Figure .The median frequency of hospital-origin BSI caused by MRSA per 100 000 patients tested for hospital-origin BSI was 132 (range 0–857), by 3GCREC was 1269 (range 427–2877), by 3GCRKP was 2363 (range 0–8045), by CREC was 129 (range 0–1204), by CRKP was 1306 (range 0–5432), by CRPA was 447 (range 0–1331) and by CRAB was 4472 (range 1460–11 968). The median frequency of AMR BSI for 3GCRKP, CREC, CRKP and CRAB was higher in TCHs than in SCHs  and anonymous hospitals no. 16 and 25 (among SCHs) had the highest frequency Figure . We alsoWe also estimated the frequency of hospital-origin AMR BSI per 100 000 admissions and per 100 000 patient-days at risk for hospital-origin BSI (data not shown), and similar findings were observed.P > 0.10), except that the median all-cause in-hospital mortality of patients with community-origin CRKP BSI was higher in TCHs than in SCHs .The median all-cause in-hospital mortality (%) of patients with hospital-origin AMR BSI caused by MRSA was 42% (range 0%–100%), by 3GCREC was 33% (range 0%–67%), by 3GCRKP was 55% (range 24%–100%), by CREC was 50% (range 0%–100%), by CRKP was 57% (range 10%–100%) and by CRAB was 64% (range 26%–100%). Difference in the all-cause in-hospital mortality (%) of patients with AMR BSI caused by each AMR pathogen was not observed between TCHs and SCHs  nurses, 7 pharmacists, 7 information technology staff, and 2 medical doctors participated in the training and provided feedback. All microbiology laboratory staff responded that the cumulative antibiograms obtained from the AMASS would be useful for their reports to their AMS and IPC teams and to national authorities. All medical doctors, IC nurses and pharmacists agreed that the summary data reported by the AMASS could be used to support their AMS and IPC programmes.This study demonstrates the utility of local and timely AMR data generated by the AMASS for local and national actions in the real-world setting. The utility of the data is shown by the ability to compare epidemiology data of participating hospitals in a timely manner and by feedback from the participating hospitals. The MoPH Thailand is inviting all 127 public TCHs and SCHs to utilize the AMASS in 2023 and planning to update the AMR dashboard by using data generated from the AMASS nationwide.The AMASS empowers hospitals to analyse, generate and share their own cumulative antibiograms, together with their total number of patients and deaths with AMR BSI, in a quick manner (i.e. within 2 months after the end of the year) and with minimal resources required . The AMR data obtained highlight the significant burden of AMR infections in participating hospitals in 2022 and the urgency to step up actions against AMR infections countrywide.10 and IPC programmes.11 The observed numbers of patients and deaths with AMR BSI in their own hospitals last year can be used to communicate with all levels of healthcare workers, including the directors and executive boards of the hospitals, better than the modelling data, national estimates or global estimates.16 Some participating hospitals are generating their own AMR reports independently using the data from previous years , discussing the findings among staff in their own hospitals, and sharing those reports with the MoPH.At the local hospital level, the frequency of hospital-acquired AMR BSI and other data of patient outcomes  can be used as measures/indicators to monitor and guide their AMSAt the national level, the ability to obtain and compare reliable AMR data between hospitals in a quick manner is crucial for designing interventions and resource allocation. If the AMASS were utilized by all TCHs and SCHs in Thailand, the policymaker could understand the situation of the AMR in the country, in each region and in each hospital much more clearly. Utilization of the AMASS can also support participating hospitals to join the WHO GLASS Thailand.9 the National Drug Policy and National Drug System Development Strategy,17 the Rational Drug Use Service Plan,18 and the national plan to achieve the sustainable development goals (SDGs) in Thailand.19The future version of the AMASS aims to include automatic analysis of electronic drug prescription data and generate reports on diagnostic and antibiotic use practices. The availability of data of AMR burden, diagnostic practice and antibiotic use practice at the local hospital level nationwide will support the national action plan on AMR,20 The all-cause in-hospital mortality reported by the AMASS21 can be used by modelling studies together with studies using recommended methods for estimating attributable mortality of AMR BSI.22–25At the international level, the data from hospitals of all sizes in LMICs can be helpful for modelling studies estimating the global burden of AMR.8 The frequency observed in this study is relatively higher than that previously observed.8 The change of the AMR burden over time will be studied separately using the data of 127 public TCHs and SCHs in 2022.The higher frequency of hospital-origin AMR BSI in TCHs compared with SCHs observed in this study is consistent with the previous study using the data of 49 hospitals from 2012 to 2015.Escherichia coli, E. coli, ESBL E. coli and ESBL-producing E. coli). For AMASS users, we emphasize the importance of completing the dictionary files and validating the AMR reports with log files, as well as comparing the data obtained in the report with their previously known workload , their previous AMR reports (if available) and manual data calculation.26The AMASS has some common issues that users face. Firstly, users need to change the Windows region settings from Thailand to either US or UK because the AMASS cannot run properly with the Windows region setting as Thailand. Secondly, dates must not be exported as date time variables. Users need to export dates as date variables or generate new variables in the Excel files as date variables prior to running the AMASS. Lastly, users need to complete the dictionary files using the log files as guidance. The completion of the dictionary files is crucial because it is not uncommon for each bacterial species to be recorded in the LIS with multiple names  reported in this study was all-cause in-hospital mortality, and could be much lower than the true all-cause mortality because a preference to die at home is high in some regions in Thailand.27 The comparisons between mortality among patients with AMR BSI, antimicrobial-susceptible BSI and ‘no infection’22–25 are not within the scope of this study. Fourth, VRE was not included in the analysis because the proportion and frequency of VRE BSI in our study hospitals were still relatively low during the study period (https://doi.org/10.6084/m9.figshare.22339933). Fifth, comparing AMR data between hospitals will need to be performed carefully due to the difference in classes, types or levels of the hospitals, case-mix, blood culture utilization and other confounding factors.29 In addition, the total number of cases and deaths with AMR BSI in each hospital depends not only on AMS and IPC practices, but also on the size of the hospital . Therefore, the frequency and mortality rate  should be calculated and used to compare the AMR burden between hospitals with similar levels of care and between areas. Sixth, formal qualitative and quantitative studies for feasibility of the AMASS and utility of the data were not performed. These will be conducted in the next phase of the AMASS implementation.The study has some limitations. First, the AMASS requires users to validate the autogenerated reports as recommended for any analytical software.In conclusion, local and timely AMR data are crucial for local and national actions. Empowering local hospitals to analyse, validate and utilize their own AMR data in a timely manner and with minimal effort, by using analytical software such as the AMASS, is strongly encouraged.dlad088_Supplementary_DataClick here for additional data file.


Bacillus cereus) in antimicrobial susceptibility test. As a result of these findings, cyclotide-containing nanofibers with antimicrobial activity can be produced for pharmaceutical research and development studies.In this study, antimicrobial nanofibers were produced with the mixtures of polyvinyl alcohol (PVA) and cyclotide-rich fractions by electrospinning method. After extraction, the first separation was carried out with C18 flash chromatography and then fractioned into five separate parts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The molecular weights of cyclotides in each fraction were determined by quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS). Cyclotide-rich fractions were mixed with 10% of PVA solution and nanofibers were produced from this biocomposite mixture by electrospinning method. The nanofibers were characterized by field emission scanning electron microscopy (FE-SEM), and it was observed that 100% peptide-containing nanofibers  had more regular fiber textures. The presence of the peptides in the nanofiber was also confirmed by analytical RP-HPLC, as the peptides in both peptide fractions and nanofiber solutions have the same retention times. The nanofibers produced with the fourth cyclotide-rich fraction showed activity against gram-positive bacteria ( HPLC chromatograms were recorded with a UV detector at 215 and 280 nm, and the fractions were collected into different containers for every 10 min during the 60 min . Only Fractions 2, 3, and 4 were used as there is enough peptide material for further studies (>5 mg).The flower part of the plant (perature . After f2O:0.1% FA (v/v) and Solvent F: 90% ACN:10% H2O:0.5% FA (v/v/v)]. The column temperature was adjusted to 25 °C. The mass spectra were recorded in positive ion mode and the mass/charge ratio was set to 500–3000 Da. LC-MS chromatograms and spectra were analyzed by Agilent Qualitative Analysis B.06.00 software.The cyclotide content of the collected fractions were then characterized by liquid chromatography-mass spectrometry (LC-MS). The LC-MS analyses of the samples were performed with a quadrupole time-of-flight liquid chromatography-mass spectrometer (Agilent G6530B Q-TOF LC-MS) equipped with a dual AJS ESI source. Nitrogen was used as both drying gas with 13 L/min flow rate and as nebulizer with 35 psi pressure. The nebulizing gas temperature was set at 325 °C, capillary potential 4000 V and the sheath gas flow rate was 12 L/min. Fragmentor voltage was set to 165 V and the skimmer voltage was 65 V of the MS TOF analyzer. One-milligram/milliliter sample solutions of Fractions 2, 3, and 4 were prepared in Solvent E and 50 μL of each solution was injected and the peptides were separated on an Agilent Poroshell 120 SB-C18 column . A 2% Solvent F gradient method with a flow rate of 0.3 mL/min for 60 min was used [Solvent E: H2O were electrospun using an electrospinning system  equipped with a 5 mL of 0.8 × 38 mm 21G sterile syringe and a fiber collector rotary plate . PVA was dissolved in distilled water and stirred in an ultrasonic bath at 80 °C for 2 h to ensure a complete dissolution. After PVA was completely dissolved in water, it was left to stir with a magnetic stirrer at room temperature over 24 h. The distance between the syringe needle tip and the collector was adjusted to 10 cm and the fibers were produced at 3.5 mL/h flow rate with 24 kV applied voltage. Based on the FE-SEM images, the optimum polymer concentration was determined as 10% PVA in H2O (w/v) as it has the better fiber texture and diameter compared to other nanofibers produced with different parameters.To determine the appropriate PVA percentage, polymer solutions having 5, 10, and 15 wt % of PVA in double distilled H.Firstly, 6.25 mg, 12.5 mg, 25.0 mg, and 37.5 mg of cyclotide mixtures from the second, third, and fourth fractions were mixed separately with 10% PVA solution to prepare 25, 50, 100, and 150 wt % of cyclotide-rich fraction/PVA mixtures, respectively. The solutions were stirred with a magnetic stirrer for 4 h at 20 ± 2 °C. Twelve cyclotide-rich fraction/PVA nanofibers in total were produced using the same parameters that was used to produce PVA nanofibers. The produced nanofibers are named as NFX-Y, where X is the fraction number and Y is the percentage of cyclotide-rich fraction in 10% PVA solution (w/v) .Morphological characterization of nanofibers was performed with FEI Nova NanoSEM 650 Field Emission Scanning Electron Microscope (FE-SEM). Before the images were obtained by electron microscopy, samples were covered with gold and examined with FE-SEM at a voltage of 10 kV.2 sample was cut from 100 wt % of cyclotide-rich fraction/PVA nanofiber mats  and from the nanofiber prepared only with PVA. One-milligram/milliliter solutions were also prepared from the second, third, and fourth RP-HPLC fractions. For the analysis, a method with a flow rate of 0.3 mL/min and a gradient of 2% Solution D was used for 35 min. The retention times of peptides and PVA of the nanofibers were compared to the retention times of peptides of the three RP-HPLC fractions.The presence of peptides in nanofibers were confirmed by comparing the retention times of peptides in nanofiber samples and peptide fractions alone by analytical RP-HPLC. For this purpose, 1 cmStaphylococcus aureus (ATCC 25923), Enterococcus faecium (RSKK 623), Enterococcus casseliflavus (ATCC 700327), Salmonella Typhimurium (ATCC 14028), E. coli (ATCC 25922) and Bacillus cereus (ATCC 13061). Bacterial suspensions (108 cfu/mL) were prepared from pure cultures grown on blood agar medium according to McFarland turbidity 0.5. From the prepared bacterial suspensions, inoculum was spread on the Müller Hinton agar plates. Nanofibers were cut in square pieces (approximately 1 cm2 in diameter) and then placed on the agars with inoculum. The prepared agar plates were incubated at 37 °C for 24 h. After incubation, the inhibition zones formed around the nanofiber samples were measured with a ruler. Standard antibiotics  were also included in the assay as the control agents along with nanofiber samples.Antimicrobial activities of the nanofiber produced only with 10% PVA and the peptide containing nanofibers  were tested against a panel of gram-negative and gram-positive microorganisms with antimicrobial susceptibility test. Bacterial cultures were In The dried amount of the second fraction is 0.91 g, third fraction is 0.13 g, and fourth fraction is 0.19 g. Fractions 1 and 5 were not used as there was not enough peptides for further studies (<5 mg). After RP-HPLC, the molecular weights of cyclotides in Fractions 2, 3, and 4 were characterized by Q-TOF LC-MS. In Morphological characterization of nanofibers was performed with FE-SEM. In In S. Typhimurium, E. coli, S. aureus, E. faecium, E. casseliflavus and B. cereus cultures were examined with antimicrobial susceptibility test. The nanofiber produced only with 10% PVA solution did not show any antimicrobial activity against all bacterial cultures tested. The results showed that the cyclotide-containing nanofibers also did not show any antimicrobial activity except the nanofibers produced with Fraction 4. While there was no activity observed for the nanofiber mats of Fractions 2 and 3 against B. cereus, the nanofibers produced with Fraction 4/10% PVA  showed antimicrobial activity towards B. cereus. Furthermore, as in Antimicrobial activities of nanofibers on V. biflora, one of the members of the largest plant sources of cyclotides, Violaceae [2O/FA , 20 mM sodium acetate (pH 5.0), 5 mM ammonium bicarbonate (pH 8.0) and the boiling water, respectively. The second extraction solution containing ACN/H2O/FA  resulted in higher crude extract yield and peptide content compared to other four methods. In Cybase (http://cybase.org.au/), a database that provides information about cyclic peptides (including cyclotides), there are nine wild type cyclotides from V. biflora have been reported [V. biflora were found to match these data. Also, different cyclotide masses not found in Cybase were also observed for the first time in this study. According to the RP-HPLC and mass spectrometry results, the V. biflora plant is a rich source of cyclotides. As reported in previous studies, the number of cyclotides obtained in each fraction is different from the literature because of the possible differences of expression levels or identities of cyclotides in plants can be vary depending on the season that the plant harvested and the plant parts used for the extraction studies [In this study, we have used cyclic peptides (i.e. cyclotides) and PVA to produce nanofibers with antimicrobial activities. Cyclotides were extracted from iolaceae . In ordeiolaceae . In thatreported . In this studies ,38.After fractionation and characterization of cyclotide-rich fractions, nanofibers were produced by electrospinning. The distance between the needle tip and the collector plate was set to 15 cm at first. However, since the fiber formed in the collecting plate did not have a proper morphology and accumulation, the distance was updated as 10 cm. It has been observed that the fiber reaches and accumulates much better at this distance which is consistent with the results reported previously in which after determination of the appropriate parameters for electrospinning, 10 cm collector distance was used for the production of nanofibers of cathelicidin peptide LL37 with polyethylene oxide (PEO) and Magainin II peptide with poly(lactide-co-glycolide) (PLGA) or PLGA/gelatin mixtures , 40. MagUnlike these last two studies, instead of covalent immobilization, we mixed cyclotide fractions with PVA first as in the production of antimicrobial nanofibers of pegylated LL-37 peptide (PEG-LL-37) with polyethylene oxide. Different concentrations of PEG-LL-37 peptide were used to examine the elimination of bacterial colonies at different rates. In our study, appropriate amount of peptide fractions was mixed with 10 wt % of PVA solution to prepare 25, 50, 100, 150, and 200 wt % of cyclotide-rich fraction/PVA mixtures. However, 200% w/v cyclotide-rich fraction/PVA mixture could not be prepared as this amount of peptide did not mix homogeneously with PVA solution which could be the result of the hydrophobic nature of cyclotides. In addition to the needle distance and the amount of active compound mixed with the polymer, the nanofiber diameters are also consistent with the literature values. The nanofiber diameters were reported 100–500 nm for PEG-LL-37 peptide-PEO nanofibers, 350 nm for Cys-KR12 peptide SF nanofibers, 715 nm for Magainin II peptide PLGA nanofibers, 356 nm tigecycline-loaded sericin/PVA composite fibers ,31,39,40V. odorata Linn. and V. tricolor herb extracts obtained in solvents with different polarity were tested on different microorganism including especially P. aeruginosa, S. aureus and C. albicans [V. odorata methanol extracts on S. aureus was 16 mm and 13 mm for P. aeruginosa [V. tricolor have 0.15 mg/mL and 1.25 mg/mL minimum inhibitory concentration (MIC) values on both S. aureus and B. cereus, respectively [E. coli and P. aeruginosa, both ethanol and methanol extracts have 2.50 mg/mL and 1.25 mg/mL MIC values, respectively. These results show that the Violaceae plant extracts have antimicrobial activities on microorganisms that we have used in this study as well. However, instead of using the whole extracts for antimicrobial studies, we first separated the cyclotide-rich fractions and fractionated again into different parts. Then, three of these cyclotide-rich parts were used to prepare nanofibers with antimicrobial activities. The nanofibers produced with pegylated LL-37 peptide and PEO, the antimicrobial activities of these nanofibers were tested by disk diffusion method with E. coli and it is found that the antimicrobial activity of LL37 can be maintained on the nanofiber mat [B. cereus. Based on the previous studies, cyclotides with antibacterial activity can be listed as cycloviolacin O2 (cyO2), circulin A and circulin B (cirA and cir B), kalata B1, cyclopsychotride and cyclotides HB10 and HB11 from Hedyotis biflora [Klebsiella pneumoniae, Pseudomonas aeruginosa, S. enterica serovar Typhimurium LT2 and E. coli. However, the cyO2 was inactive against S. aureus and E. faecium [K. pneumoniae, which attributed to the high strain variability among the bacterial species. However, they also reported the antibacterial activity of cyO2 on A. baumannii for the first time [In previous studies, the antimicrobial activities of albicans –43. In oruginosa . In anotectively . For theiber mat . In this biflora ,44,45. IB. cereus) in antimicrobial susceptibility test. As a result of these findings, cyclotide-containing nanofibers with antimicrobial activity can be produced for pharmaceutical research and development studies. It is also possible that the antibacterial activity is increased as the amount of peptide in the nanofiber increase.In conclusion, Viola plant family is a rich source of cyclotides, and the valuable properties of these peptides can be harnessed by using nanofibers in addition to grafting studies where the cyclotide framework is used as a stable scaffold for drug design and development. The nanofibers showed activity against gram-positive bacteria (


Postharvest biology of horticultural crops is a scientific discipline of the biological processes that occur in horticultural crops after harvest. It is the foundation for practical strategies and technology for prolonging the postharvest longevity of produce. The postharvest longevity includes storage, logistics and transportation, and shelf life. It is estimated that up to 70% of produce are lost after harvest. Understanding of postharvest biological processes will allow us to devise ways to control these processes for improving the produce quality (and quantity) and reducing the economic loss.Molecular Postharvest Biology” to reflect the advances.Molecular biology and related techniques have been increasingly used in almost all aspects of horticulture research including postharvest biological studies  and related biological processes. It also discusses novel strategies and technologies for delaying senescence that have been derived from the molecular understanding of the senescence processes. Future directions are also presented. AtNAP is one of the most important transcription factors discussed in the Review. Two research articles in the collection report the most recent findings that AtNAP promotes leaf senescence by activating the gene of cytokinin oxidase 3 (that degrades senescence-inhibiting plant hormone cytokinins)  are predominantly fungi  that render food (and feed) unsafe and contribute to significant loss of produce. Molecular understanding of pathogens and pathogenesis will help control the postharvest diseases. The special collection proudly presents a review article entitled “Molecular basis of pathogenesis of postharvest pathogenic fungi and control strategy in fruits: progress and prospect” (Zhang et al. In summary, the special collection on “Molecular Postharvest Biology” with mix of review and research articles represents fore frontiers in this field, and will be excellent references for researchers and a textbook for advanced trainees.


Frontiers in Oncology is dedicated to the current trends in the diagnosis and treatment of hybrid or mixed myelodysplastic/myeloproliferative disorders. This group of myeloid neoplasms has not yet received enough attention in the various classifications proposed for these diseases, for several decades, been placed in the past under the umbrella of myelodysplastic syndromes (MDS) or that of myeloproliferative neoplasms (MPN).This Research Topic in However, it has ultimately become clear that hybrid or mixed MDS/MPN have a profile of clinical, hematologic, cytomorphologic, histomorphologic, pathogenetic, cytogenetic, and molecular characteristics, indicating that enlisting or classifying them among other disease entities, such as the typical and clearly identifiable MPN or MDS, may not be appropriate or even useful and practical. It should be emphasized that all the articles included in this Research Topic have been submitted, revised, and published before the recently published fifth edition of the World Health Organization (WHO) Classification of Myeloid Neoplasms. Thus, the authors have followed the previous fourth edition of WHO Hybrid or Mixed MDS/MPN, published in 2016, according to which five disease entities were included, namely, atypical BCR-ABL negative chronic myelogenous leukemia (aCML), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), MDS/MPN unclassifiable (MDS/MPN-U), and MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RST) as a provisional entity  with SF3B1 mutations. A new entity, namely myeloproliferative neoplasm with PCM1-JAK2 rearrangement, has been proposed, whereas JMML has been removed from this category  are characterized by a variety of chromosomal and, particularly, of molecular alterations, such as somatic mutations. Moreover, none of these somatic mutations, or any combination of them, nor any chromosomal aberration is pathognomonic for the entire group of mixed MDS/MPN or even for a single member-entity of the group. An additional logistic problem is that these are very rare and inhomogeneous diseases, and therefore, large databases and biobanks, which could potentially allow us to better analyze the disease biology, are not easily available , 5.Frontiers in Oncology has therefore asked experienced hematologists, hematopathologists, and geneticists to report on the epidemiology, pathogenesis, diagnosis, classification, prognosis, and therapeutic approaches of mixed MDS/MPN. In this Research Topic, Barone et al. report that in patients with polycythemia vera (PV), the circulating megakaryocyte-derived extracellular vesicles (EV) were significantly decreased, whereas platelet-derived EVs were increased and that PV patients also had an abnormal microbial DNA signature, which can generate an abnormal inflammatory network, potentially favoring the pathogenesis of the disease. In a review, Diamantopoulos and Viniou summarize the state-of-the-art diagnostic procedures, the recently-described molecular characterization of the disease, i.e., the recurrent pathogenetic mutations, and the current treatment approaches for patients with aCML. A comprehensive review of the complex and heterogeneous molecular pathogenesis and pathophysiology of CMML, with the application of the currently available research technologies, as well as the development of potential molecular targets for the design of novel therapies is presented in an excellent article by Geissler. The rare entity of myeloproliferative neoplasm with PCM1-JAK2 rearrangement is reviewed by Sun et al., following the description of two new cases. The authors discuss the poor prognosis and the lack of treatment guidelines for this entity, as well as the unpredictable and heterogeneous response pattern of treatment with ruxolitinib. They suggest that the combination of ruxolitinib and pegylated interferon might be more effective. Fontana et al. add molecular pathogenetic considerations on BCR/ABL-negative aCML, and discuss the influence of the molecular profile of the disease on prognosis and on the potential treatment options, including the novel and emerging targeted treatments. Küendgen et al. compile the epidemiological characteristics of hybrid or mixed MDS/MPN and emphasize that with the exception of the most common disease, namely CMML, there is a lack of reliable and convincing information, mainly attributed to the rarity of these syndromes, but also to the changing classification systems and the blurry and vague limits of each of them. Liapis et al. discuss the various options for first-line treatment available to patients with advanced CMML and analyze the existing data of the two main treatment approaches, namely, cytotoxic treatment and hypomethylated agents, and how this kind of treatment can be used as a bridge for allogeneic stem cell transplantation in transplant-eligible patients. Symeonidis et al. present all the published data from a few prospective studies but mainly from retrospective studies on allogeneic stem cell transplantation for mixed MDS/MPN and analyze the prognostic factors for the various transplantation outcomes. Finally, Thomopoulos et al. complete the round of articles with an overview of the clinical characteristics, molecular genetics, and the existing and emerging therapeutic approaches for patients with chronic neutrophilic leukemia, a very rare disease with several unmet needs for appropriate diagnosis and effective treatment.This Research Topic in In summary, this Research Topic consists of a collection of excellent articles, aiming to help interested readers to navigate through the classifications, understand the complex pathogenesis a little bit more, and get inspiration with regard to therapeutic considerations. We believe that the readers will enjoy reading this Research Topic.AS and UG have equally contributed in the design and scientific supervision of the content of this Research Topic, and they also have both contributed to the writing of this Editorial. All authors contributed to the article and approved the submitted version.


Condition-based molecular generation can generate a large number of molecules with particular properties, expanding the virtual drug screening library, and accelerating the process of drug discovery. In this study, we combined a molecular graph structure and sequential representations using a generative pretrained transformer (GPT) architecture for generating molecules conditionally. The incorporation of graph structure information facilitated a better comprehension of molecular topological features, and the augmentation of a sequential contextual understanding of GPT architecture facilitated molecular generation. The experiments indicate that our model efficiently produces molecules with the desired properties, with valid and unique metrics that are close to 100%. Faced with the typical task of generating molecules based on a scaffold in drug discovery, our model is able to preserve scaffold information and generate molecules with low similarity and specified properties. Drug discovery and development constitute a complex and challenging process demanding extensive time, resources, and domain expertise ,2,3,4. TMolecular generation models based on artificial intelligence (AI) have showcased immense potential and have become pivotal in de novo drug design ,12. ThesIn this work, we focus on the molecular generation task to produce molecules with designated properties, thereby expanding molecular screening libraries . We propTo test the ability of our model to generate molecules with a specific property, we specified a single property (Synthetic Accessibility Score (SAS)) , QuantitAs shown in In the context of generating a single property, our findings demonstrate  that theConcurrently, we tested the performance of molecular generation based on multi properties. In summary, although MolGPT and our model perform similarly in terms of validity, uniqueness, and novelty, our model is superior in terms of the stability of the results, i.e., the generated samples are more consistent and reliable across multiple experiments. It can be concluded that our model is better in this molecular generation task, and the generated molecular structures are not only of high quality but also show more stable performance in different specified properties experiments.The molecular scaffold constitutes a pivotal element in drug design, influencing the structure, properties, and interactions of molecules. By strategically designing and modifying the molecular scaffold, drug molecules can attain specific biological activities, pharmacokinetics, and safety profiles, thereby laying a robust foundation for novel drug development. Therefore, we tested the performance of GraphGPT in generating molecules for a given scaffold and properties.We adopted the five molecular scaffolds used by Bagal et al. , and tesWe visualize the properties of the generated samples in the scaffold and single property-based molecular generation experiments. As can be seen in We conducted unconditional molecular generation on the GuacaMol  dataset,As shown in We employed the model trained on the Moses dataset for molecular generation, predefining the molecular scaffold, logP, and TPSA before generating molecules. Five molecular scaffolds were used to test the performance of molecular optimization. LogP was set to 2.0, and TPSA was set to 40.0. In other words, we aimed to generate molecules that retained their molecular scaffolds while achieving logP and TPSA values close to 2.0 and 40.0, respectively. Partially sampled molecules are illustrated in To verify the effect of different decoder layers as well as graph encoders on miniGPT, we performed ablation experiments. As shown in We use a GAT-based graph encoder to encode the molecular structure in order to solve the problem of missing topology in the sequence-based molecular generation process. The alignment of the graph encoder with the sequence encoder enables the fusion of structural information into the sequence encoder. Experiments demonstrate that the model is able to generate more accurate molecular properties with little degradation in performance such as molecular validity. There are also variants of SMILES such as SELFIES , R-SMILEIn this section, the overarching architecture of the model is initially presented. Subsequently, the structure of the graph-based encoder is expounded upon. Following this, the encoding of molecular scaffolds, properties, and molecular SMILES sequences are delineated through the utilization of a sequence encoder akin to the GPT framework. Lastly, the employed loss function is elucidated, facilitating the realization of molecular sequence generation fortified by graph-enhanced structures.A sequence-to-sequence molecular generation model enhanced with graph structures was proposed. This model was capable of generating molecules based on molecular scaffolds and properties. As illustrated in To address the deficiency in capturing the previous molecular topology within the molecular generation model, we introduced the Graph Attention Mechanism (GAT)  for encojh signifies the feature representation of atom j, W denotes the transformation matrix, and || denotes the concatenation of the features of atom i and atom j. Following the application of the LeakyReLU activation function, the resultant i,je emerged, representing the weight indicative of the influence of atom j on atom i. Equation (2) encapsulated the process of aggregating all connected atoms to atom i and subsequently normalizing the aggregated values. Incorporating a multi-head attention mechanism, as depicted in Equation (3), the elevated-dimensional feature representation of atom i was updated. The Sigmoid function (σ) was employed as the activation function, yielding the refined atom feature ′jh, which was subject to the dropout function. Following the passage of the molecule through the two layers of graph attention networks, a ReLU activation function was applied, succeeded by a global maximum pooling operation. It is pertinent to note that our graph encoder was exclusively operational during the training phase; during inference, its functionality was suspended due to the unavailability of molecular graph structural information.We commenced by employing one-hot encoding to represent atomic attributes, encompassing atom type, degree, amount of hydrogen, and implicit valence. Subsequently, the interatomic relationships within the molecule were established, employing a two-layer graph attention for the encoding of graph structure. As expressed in Equation (1), the one-hot tensor of each atom was subjected to a linear transformation, projecting it into a higher-dimensional space. Here, The molecules were portrayed as sequences in the form of SMILES, serving as the input for the model. Additionally, RDKit  was utilQ, K, and V represent the Query, Key, and Value vectors, respectively, T represents the transpose, and kd represents the Key vector dimension. After the sequence representation through the class GPT, the representation of the sequence is mapped to the same space as the representation of the graph encoder through a mapper, which is used with a fully connected representation in this study.In fact, our model employed a decoder module akin to the Transformer architecture, comprising 8 stacked decoders: a design reminiscent of architectures found within the GPT series. A comparison between the model we utilized and GPT-1 is provided in BTL, encapsulates the disparity between the molecular representation post-traversal through the graph encoder and SMILES sequence encoder. Given that these representations emanate from differing perspectives in molecular characterization, the features resulting from the graph encoder and the sequential encoder should exhibit close proximity. We constrained sequential encoding by employing the graph-encoded representation, thereby enabling the model to indirectly glean topological structural information inherent to the molecule. The second loss function, denoted as groundL, encapsulates the divergence between the molecular predictions yielded by the model and the actual molecular structures. As depicted in Equation (6), the computation approach for measuring the gap between the graph structure encoding and the sequence encoding adopts the Barlow Twins loss function [λ serves as a hyperparameter, set to 0.005, and ijC represents the correlation coefficient between the graph structure encoding and the sequence encoding, as calculated in Equation (7). Within these equations, b denotes a batch index, while i and j, respectively, index the graph encoder and sequence encoder. A and B correspond to the graph encoder and sequence encoder, respectively.In order to fuse the topological information, we employ a GAT in the molecule generation task and subsequently generate molecules with specific properties. As demonstrated in Equation (5), our formulated loss function encompasses two distinct components. The first loss function, denoted as function . Here, λWe tested GraphGPT for the molecular generation task using two datasets: the GuacaMol dataset and the MOSES dataset. The GuacaMol dataset comprises a subset of 1.6 million molecules from the ChEMBL 24 database . The molValid: Valid pertains to the valid portions within the generated molecules based on SMILES syntax and atomic valency rules. We consider a molecule valid when the generated SMILES can be analyzed using an RDKit. A high valid score indicates that the model has learned the accurate representation of molecules and their chemical properties.Unique: Unique specifies that it is a case of duplicates in the generated molecule. If the newly generated molecule has not been generated before, then it is considered ideal. A lower uniqueness score suggests that the model is generating repetitive or redundant molecules.Novelty: Novelty refers to the segments present in the generated valid and unique molecules that are absent in the training dataset. This metric is employed to determine whether the model is overfitting, signifying that it has memorized the training data without generalizing to unseen molecules.s1 and s2 denote two molecules, and T represents Tanimoto similarity [S). The parameter p can be either 1 or 2.Internal Diversity (IntDivp): Internal Diversity evaluates the similarity between generated molecules. As shown in Equation (8), milarity . This enGμ is the mean and ΣG is the covariance of the distribution G. In the same way as Bagal et al. [FCD.Frechet ChemNet Distance (FCD): This metric tests the similarity of the generated molecular data to the training molecular data. As shown in Equation (9), where l et al.  for the k represents the kth properties.KL Divergence: KL Divergence was computed using a plethora of physicochemical descriptors for both the generated molecules and the training set. Lower values denote a proficient learning of the distribution of these properties by the model. The calculation is shown in Equations (10) and (11). Here, We used six metrics to assess the effectiveness as well as the diversity of generated molecules by the model, and here is what the metrics mean:We juxtaposed our model against seven distinct baseline models, encompassing CharRNN , VAE 5050, AAE , JTN-VAEThe primary contribution of this work lies in the successful integration of graph structures into an NLP-inspired sequence-to-sequence framework. This novel approach not only harnesses the power of language modeling but also seamlessly captures and exploits the topological features of molecules. Our model demonstrates exceptional performance in generating molecules with higher validity, uniqueness, and diversity. Unconditional molecule generation, property molecule generation, and scaffold-based molecule generation experiments all demonstrate the performance of our model. Through extensive experimentation and evaluation of established datasets, we showcase GraphGPT making significant strides in achieving more advanced structure-aware molecular generation techniques. Our method facilitates the construction of large-scale molecular screening libraries and the generation of lead compounds with specified properties, thereby propelling advancements in AI-driven drug discovery and related fields.


Kaposi's sarcoma (KS) is a rare multifocal angiogenic tumor often seen in immunocompromised setting such as acquired immunodeficiency syndrome (AIDS) or organ transplantation recipients. Pemphigus vulgaris (PV) is a rare blistering disorder with mucocutaneous involvement for which immunosuppressive therapy has long been the core of treatment. Iatrogenic form of KS has been reported infrequently in pemphigus patients as a result of long‐term immunosuppressive therapy.We describe a 39‐year‐old male patient with confirmed diagnosis of PV who developed KS after receiving immunosuppressive agents for his pemphigus. KS was initially localized to the oral cavity with features mimicking exacerbation of his pemphigus.This interesting case of KS suggests that dermatologists visiting patients with pemphigus with discomfort in the oral cavity should have a high degree of awareness and consider other differential diagnoses along with merely an exacerbation of PV. Histopathologic and DIF examinations stablished the diagnosis of PV. His disease had initially been controlled with 60 mg/day prednisolone which tapered to 30 mg/day during 1 month and rituximab (one cycle consisting of one infusion of 500 mg every week for 4 weeks) was added to his therapeutic regimen leading to a considerable clinical improvement. Hence, prednisolone was rapidly tapered to 10 mg/day in 3 months because of his cushingoid appearance and also achieving partial clinical remission. The Pemphigus Disease Area Index (PDAI) score of cutaneous and mucosal lesions from 11 and 10 at the baseline, declined to 0 and 3 at the fifth month of treatment, respectively.The patient had also taken Isoniazide (INH) and vitamin B6 as tuberculosis prophylaxis due to positive skin tuberculin test (induration: 13 mm). Patient was otherwise healthy.By the time, the prednisolone dosage was lowered to 7.5 mg/day and cutaneous PDAI score was 0, but he was still complaining of a persistent odynophagia and dysphonia.On physical examination, the patient had generally good health and no remarkable finding was noted except for mild sore throat and an erythematous nodule with erosive surface at the end of lingual area Figure . AlthougMRI of cervical soft tissue revealed an isosignal 23 × 73 mm mass at oropharyngeal region on epiglottis which was enhanced after contrast injection. Pressure effect on airway was also noted.The patient was referred to the Otolaryngology Department, where the nodule was totally excised after a laryngoscopic evaluation in which a large mass was detected at the base of tongue Figure . HistopaDuring systemic evaluation, no history of opportunistic infections, B symptoms or any sign or symptom regarding visceral involvement of KS was noted.Taking into consideration the clinical complete remission of PV in patient as well as the low values of anti‐Dsg1, 3 , we decided to taper off the prednisolone.One month later, two other nodular violaceous lesions on his cheek and ankle were appeared Figure  which weAny occurrence of KS lesion has not been observed until now (21 months after excision of the last lesion) and the patient did not describe any relapses of PV or KS lesions.3KS is an angioproliferative neoplasm of low‐grade malignant potential.It is hypothesized that altered immunity weakens the immunological surveillance and leads to reactivation of a latent HHV‐8 infection in genetically susceptible patients.KS is classified into four types: classic, endemic, immunosuppression‐associated or iatrogenic and AIDS‐associated or epidemic.Association of pemphigus (mainly PV) and KS has been previously reported.Iatrogenic KS often has similar clinical features to the AIDS‐associated KS including fulminant course with widespread lesions, tendency to visceral involvementAs mentioned before, most of the cases of KS arises in immunocompromised patients have an aggressive and fulminant course, whereas the relatively fair course and good response to treatment in our case might be in favor of the fact that the appearance of KS was not secondary to immunosuppression. It is worthy to note that occurrence of the second and third KS lesions in our case were 1 month after tapering off the prednisolone.Altered immunity due to receiving immunosuppressive agents is not the only explanation for association of PV and KS. Although not proven yet, HHV‐8 has been introduced as a triggering factor of bullous disease, especially PVResults of the recent cohort study showed that half of pemphigus‐associated KS cases developed their KS lesions before the start of immunosuppressive therapy that is fairly in favor of the role of HHV‐8 infection as a triggering factor in patients with pemphigus.HHV‐8 detection in tissue samples of our patient could have shed some light on this matter of debate and suggested the pivotal role of HHV‐8 at least, in PV‐associated cases with a relatively fair course of disease, but regretfully, we could not test it which is a major limitation of this study.The fact that tuberculin test in our patient was positive without any clinical manifestation of tuberculosis, might be inferred as the relative strength of cell‐mediated immunity that resulted in a benign course of KS in our case.Treatment of KS depends on the clinical presentation, extent of involvement and associated comorbidities. In local diseases, radiation, photodynamic therapy, intralesional vinblastine, topical alitretinoin gel, cryotherapy, curettage and electrodesiccation are main therapeutic options. For extensive disease, a combination of surgery, chemotherapy and radiation is advocated. Some recent therapeutic agents such as imatinib and matrix metalloproteinase inhibitors are also being triedIn conclusion, we reported a case of KS with an unusual presentation to emphasize the fact that careful examination and clinical suspicion are the key factors for correct diagnosis. In fact, throat burning and odynophagia in PV patients might not be merely due to pemphigus erosions and should raise the suspicion of occurrence of other pathologic processes such as KS even in HIV‐negative patients.Furthermore, we highlighted the fact that not all of patients with pemphigus‐associated KS have fulminant course. Therefore, the crucial question to be resolved is how we can discriminate between steroid‐associated KS and disease‐associated KS in patients with pemphigus.Implementing some proceedings such as skin tuberculin test might be useful in answering this question and predicting the course of disease. Further studies are needed to shed more light on this topic.Kamran Balighi: Conceptualization ; supervision ; visualization . Maryam Ghiasi: Data curation ; methodology ; project administration . Zeinab Aryanian: Investigation ; writing – original draft . Zahra Nikyar: Investigation ; project administration ; writing – original draft . Parvaneh Hatami: Investigation ; visualization ; writing – review and editing .The authors declare that they have no conflict of interest regarding the publication of this article.The patient provided written informed contest to publication of this case report and accompanying images.


Mycobacterium bovis) in the United States of America occupies a position both enviable and daunting. Excepting four counties in Michigan comprising only 6109 km2 (0.06% of US land area) classified as Modified Accredited, as of April 2022 the entire country was considered Accredited Free of bTB by the US Department of Agriculture for cattle and bison. On the surface, the now well-described circumstances of endemic bTB in Michigan, where white-tailed deer (Odocoileus virginianus) serve as a free-ranging wildlife maintenance host, may appear to be the principal remaining barrier to national eradication. However, the situation there is unique in the U.S., and far-removed from the broader issues of bTB control in the remainder of the country. In Michigan, extensive surveillance for bTB in deer over the last quarter century, and regulatory measures to maximize the harvest of publicly-owned wildlife, have been implemented and sustained. Prevalence of bTB in deer has remained at a low level, although not sufficiently low to eliminate cattle herd infections. Public attitudes towards bTB, cattle and deer, and their relative importance, have been more influential in the management of the disease than any limitations of biological science. However, profound changes in the demographics and social attitudes of Michigan’s human population are underway, changes which are likely to force a critical reevaluation of the bTB control strategies thus far considered integral. In the rest of the U.S. where bTB is not self-sustaining in wildlife, changes in the scale of cattle production, coupled with both technical and non-technical issues have created their own substantial challenges. It is against this diverse backdrop that the evolution of whole genome sequencing of M. bovis has revolutionized understanding of the history and ecology of bTB in Michigan, resolved previously undiscernible epidemiological puzzles, provided insights into zoonotic transmission, and unified eradication efforts across species and agencies. We describe the current status of bTB eradication in the U.S., how circumstances and management have changed, what has been learned, and what remains more elusive than ever.Having entered into its second century, the eradication program for bovine tuberculosis (bTB, caused by  Mycobacterium bovis) in the United States of America occupies a position at once both enviable and daunting. With the exception of four counties in Michigan comprising only 6109 km2 (0.06% of US land area) which were classified as Modified Accredited (MA), as of April 2022 the entire country was considered Accredited Free (AF) of bTB by the US Department of Agriculture (USDA) for cattle and bison (Bison bison). All 50 states were classified as MA, two steps below AF, for captive cervids. Between 1 October 2018 and 30 September 2019, only 10 cattle herds were detected as infected with M. bovis; 4 of those were in Michigan. Considering where the US bTB eradication program began in 1917, with 5% of US cattle tuberculous on average ), the clades diverged relatively rapidly between about 1974 and 1987 as noted in Fig. Owing primarily to the difficulty of observing free-ranging wildlife, understanding the emergence of infectious disease and how it has spread over space and time are frequently challenging and understudied problems. Michigan’s bTB outbreak is no exception. A recent genomic analysis  of WGS f994; see , 31). NoM. bovis in the United Kingdom and New Zealand ) that supports fewer deer but is still economically valuable. In general, the focus is on building long-term trust relationships with area landowners, to increase transparency of agency decision making to the public, agency credibility, and hopefully, cooperation [The potential ramifications of these trends for bTB control efforts cannot be overstated. With strong evidence of density dependence on bTB transmission , fewer hperation .Yet trust does not necessarily entail increased participation in agency-directed management –52, and M. bovis genotypes unique to Michigan (and thus could be considered native cases), while three herds were infected with genotypes related to those circulating in North American farmed elk herds for the past few decades. These three herds were infected by movement of infected cattle into Michigan from out of state. There have also been 6 feedlots infected by movement of cattle from infected Michigan source herds. Because these feedlots are considered ‘terminal facilities’ , they are not considered in the count of infected herds by USDA. In addition, a captive bison (Bison bison) herd in Oscoda County was diagnosed in 2014, and 6 infected farmed deer and/or elk herds have also been identified, in 1997, 2006, 2009 (2) and 2021 (2). So far, all the captive wildlife herds sequenced were infected with Michigan genotypes.To date, there have been 81 bTB-infected cattle herds in Michigan since the first was diagnosed in Alpena County in 1998. Sixty-four of these (79%) were beef herds, with the remainder  dairies. The most recent positive farm was found in January 2022. Nine farms (11%) have been infected twice, and two (2.5%) three times. Seventy-eight of these farms (96%) were infected with Michigan has split state status in the USDA bTB eradication program, meaning that officially it has two bTB accreditation statuses with respect to livestock . Four ofM. bovis in free-ranging deer. That said, whole herd testing in these outlying areas has already proven efficacious, detecting an infected beef herd in Cheboygan County in 2021. At least through the end of 2023, whole herd testing of cattle  will be required in Presque Isle County, as well as portions of six other counties within 16 km of the MA zone border. Specific details of all requirements are available elsewhere [Understandably, both cattle producers and the Michigan Department of Agriculture and Rural Development (MDARD) have opposed such testing as placing onerous regulatory burdens on them, while wildlife management is not eliminating the source of lsewhere . So althMaintenance of split state status requires that MDARD, MDNR and USDA negotiate and abide by (at least ostensibly) a Memorandum of Understanding (MOU) that stipulates the responsibilities each agency will carry out in order to maintain split state status. The most recent renegotiation of the MOU was signed by all three agencies in April, 2022 . The proWith Michigan thus far unable (or unwilling) to eliminate bTB in WTD, considerable emphasis has been placed on cattle farm biosecurity . BiosecuM. bovis to cattle at specific times of year. The USDA’s APHIS Wildlife Services branch has played a pivotal role in both programs, with primary responsibility for risk assessments, verification visits, and targeted deer removal. Since 2017, 1,890 deer have been culled from 49 farms in the designated EWB area, with another 686 deer removed from other cattle farms in the bTB area. All deer are tested for bTB at MDNR’s Wildlife Disease Laboratory, and meat from test negative deer is donated to local food charities if the farm does not keep it for personal consumption. Nine (0.3%) of these deer have cultured positive for M. bovis through May 2022.However, many producers welcomed culling before it was required. It bears emphasis that this culling is not a population reduction strategy, but rather a tool to remove habituated deer using specific farm features that present a risk of indirect transmission of A principal, if unstated, justification for implementation of farm biosecurity has been that if it proved adequate to prevent herd breakdowns, more burdensome regulation by USDA would be unnecessary. Yet previous modelling  has suggM. bovis evolutionary rate of 0.37 substitutions per year [M. bovis positive.Although perhaps not to the extent seen with wildlife data, WGS of Michigan livestock isolates has also proven critical to facilitating epidemiological insights that were not possible previously. Prior to the availability of WGSs, when cattle herd breakdowns occurred in areas relatively distant from the bTB endemic area, if cattle movement records were insufficient to prove herd to herd transmission, or absent entirely, the default assumption was that the farm must have been infected by local deer. That initiated testing of local deer within a radius around the facility, testing which typically ended with no new infected deer being found. While also demanding more resources from MDNR field staff to contact landowners, collect deer for testing, etc., the ambiguous source of the infection considerably complicated policies for both cattle and deer management in the such areas. With WGSs, the ability to implicate herd to herd transmission due to cattle movement has clarified these situations. In addition, in the core outbreak area, WGS have provided insights on longevity of herd infections, and diagnostic failures. For example, when an Alcona County beef herd was first diagnosed as infected in January 2020, the breakdown was by default concluded to be due to recent infection from deer, because as an MA zone herd, it was subjected to whole herd testing annually. However, as the herd was subjected to repeated tests and additional infected animals were found, considerable genotypic diversity was documented, as noted in Fig. per year , about oOutside of Michigan, the only remaining state with endemic bTB in wildlife is Hawaii, on the island of Molokai, presumed to be maintained in feral swine , 70. TheM. bovis isolates there [The occurrence and elimination of a bTB outbreak in cattle and free-ranging WTD in Minnesota has been described elsewhere , 73. No es there , 75.Excluding Michigan and Hawaii, from 2014 through 2021, the annual number of infected cattle herds in the US has remained low among both dairies  and beef herds . Over the last two decades, the distribution of infected livestock herds by type has been 57% beef, 34% dairy and 9% farmed cervid. While the absolute number of infected herds per year is small, it is notable that, compared to Michigan, the average size of these infected herds is orders of magnitude higher.The epidemiology of bTB in the US outside of Michigan and Hawaii can perhaps be best characterized as consisting of a low level of new introductions of genotypes of Mexican or unknown origin arising in both beef and dairy cattle, with minimal subsequent herd to herd transmission. In contrast to much of the US’ bTB eradication history, it is now extremely rare to discover an infected herd that traces to an infected source herd, or any affected herds at all. Whole genome sequencing of isolates from cases detected at slaughter from 2014–2021 has, with only a single exception, revealed previously unrecorded genotypes predominantly of Mexican origin, as shown in Fig. So what might the source(s) of these new genotypes be? Several hypotheses exist. Given the low sensitivity of slaughter surveillance (assumed by USDA to be approximately 30%), it is plausible that some infected cattle are being missed at slaughter. Yet, the genotypes are overwhelmingly novel to the US, and as bTB control in Mexico has progressed markedly over the past two decades, Mexican origin bTB cases detected at slaughter have declined exponentially since 2001. Whether the speculated number of missed infections would provide sufficient foundation for these ongoing novel genotypes is debatable. Moreover, covert infections in feeder cattle do not appear to explain the pool of infections detected in mature, US-origin culled cattle. There is a possibility that those cases could be reverse-zoonotic in origin, as WGS has documented transmission of bTB from livestock workers to cattle in the US previously . How comM. bovis. Estimates of vaccine efficacy vary widely depending on the study; however in all studies, M. bovis BCG consistently decreases disease severity even though it does not necessarily prevent infection. Most of the published BCG efficacy studies have focused on measures of individual animal protection but a recent meta-analysis [M. bovis shedding in nasal secretions and milk, use of alternative diagnostic strategies to differentiate infected from vaccinated animals and the effects of vaccination on production and comorbidities will also be examined. While unlikely to directly improve detection of covertly infected cattle in the US, measures that continue to decrease incidence and persistence of bTB in Mexico will benefit both countries.Amidst this ambiguity, USDA has provided funding for pilot projects that may help identify potential solutions. Firstly, radiofrequency individual animal identification (RFID) infrastructure is being installed in Texas cattle markets that process most of the Mexican-origin feeder cattle prior to slaughter. The RFID approach has been one of the mutually praised success stories of the bTB eradication program in Michigan, and the hope is that it will facilitate traceability of imported cattle as well. That capability could prove valuable to both the US , and to Mexico (to identify disposition of their cattle). It could also provide a marketing tool aimed at consumers, showing that the beef they eat can be accounted for from ‘farm to fork’. Secondly, additional meat inspection personnel are being placed at US abattoirs that slaughter many Mexican-origin cattle. Line speed at modern facilities is so rapid (200–400 head slaughtered per hour ) that itanalysis  suggesteAs the cattle industry in the US has changed, a key tool of the bTB eradication program, herd depopulation, has become obsolete in many cases. USDA APHIS Veterinary Services (VS) budget for funding producer indemnity for condemned herds has not kept pace with growth in herd sizes, some of which are now in the 75,000 to 100,000 head range, as the industry pursues economies of scale. For example, in 2021, VS’ entire budget for indemnity was US$1 million, equivalent to about €955,000. When assessing disposition for a recent infected Wisconsin dairy of 5,000 head, if the maximum indemnity reimbursement of US$3,000  were paid for every animal in the herd, the total cost to depopulate this one herd  would have exceeded the entire annual VS indemnity budget by 15 times, despite it not even being ‘large’ by US standards. Avenues do exist for VS to solicit the US Congress for supplemental appropriations to bolster the indemnity budget. However, the current polarized and contentious political climate in the US portends a low probability of success for such requests. In addition, the legislature is unlikely to fund supplemental appropriations repeatedly, so the circumstances where such funds are sought need to be considered carefully by VS decisionmakers. Finally, the increased size of US dairy cattle herds also means that multiple loans may be required to assemble a large herd. If different banks have liens against an infected herd, decisions to depopulate are even further complicated, as ‘ownership’ of the herd is not straightforward.More generally, although regulatory flexibility is often sought by cattle producers for infected herds both in Michigan and nationwide, there are clearly tradeoffs between regulatory flexibility and regulatory efficacy. Inevitably, some consistency in regulation is necessary. If the eradication program is not actually making consistent progress towards eradication, justification for its very existence comes into question.M. bovis genotypes not previously observed in the US and ruling out herd-to-herd transmission, WGS has also facilitated evaluation of on-farm transmission at a level of detail impossible just a few years ago. Two recent herd investigations showcase this.The use of whole genome sequencing has revolutionized epidemiologic investigations at both the national and herd level. In addition to identifying M. bovis. From 75 cows found infected, WGS identified three different M. bovis genotypes – 6A, 13A and 17B1 – none of which matched the genotype found in the source herd. One cow was found infected with 6A, one with 13A and the rest with 17B1. Whole genome sequences of the cows with 17B1 showed more than 50 cows having the exact same genotype, suggesting a common source of exposure. Most of the cows infected with the 17B1 strain calved within a 2-month period. On-farm investigation suggests the exposure occurred in the calving area. The remaining 17B1 isolates showed no evidence of cow-to-cow transmission since there were no additional accumulated SNPs shared among isolates from different cows. From this analysis, the WGS data suggested at least three different exposures for each M. bovis strain and a common source exposure, possibly human, for the cows with the 17B1 isolates.The first, an 8,000 cow herd was detected as infected in 2018 during a herd test which was required because cattle had been purchased from another herd that was subsequently found infected with The second investigation was on a multi-site facility in New Mexico with more than 10,000 dairy cows and heifers. The dairy was undergoing implementation of a test and remove bTB eradication plan and had undergone multiple tests, with the number of newly infected cows decreasing with each subsequent test. On the sixth whole herd test, nine first lactation heifers were detected as infected. An on-farm investigation into the housing of these nine heifers and previously detected infected cows identified the close-up dry pen as the place of exposure. The WGS of the isolates from the infected heifers showed a common source exposure to one of the infected cows being housed in the same close-up pen.M. bovis-infected cows to shed and expose their pen mates. Because dairy cattle are immunosuppressed around the time of calving, it makes sense that infected cows may lose control of the infection and shed during this time and that non-infected cows may be more susceptible to infection.These two herd investigations, using WGS, highlight the time around calving as a risky period for M. bovis in a North American deer-cattle system. Response to the outbreak has prompted innovations such as radiofrequency identification of individual cattle and much improved farm biosecurity, and facilitated collection of epidemiological data that are unique in the world. Moreover, the geographic scope of the outbreak has remained limited to a relatively small area, and prevalence of the disease in multiple species has been constrained. However, Michigan’s often-stated goal of “eradicating bovine tuberculosis in Michigan deer” [M. bovis infection to carry on normally and avoid the costs of disease control, as the MA zone shrinks to the smallest defensible area, the number of people subject to the pains of bTB shrinks, arguably to the point that their political clout is no longer sufficient to mount any meaningful progress towards eradication. It also magnifies the potential influence of non-cooperators [Since bTB was first diagnosed in Michigan in 1975, a great deal has been learned about the pathogenesis and ecology of an deer”  remains perators . While rIn the rest of the US, it has been argued by both cattle producers and agency staff alike that the current nationwide bTB regulatory structure is no longer likely to result in eradication. But with current weaknesses in the regulatory system as yet poorly defined, how the US should restructure the eradication program to best address them and move forward is also unclear.


A 42-year-old male with a history of human immunodeficiency virus (HIV) infection, a CD4 cell count of >400/μL, and an undetectable viral load for six months presented with a tumoral lesion on his lower limb accompanied by satellite normochromic nodules . A skin KS is a type of vascular sarcoma. In the 1980s, KS was one of the first opportunistic diseases reported in connection with HIV infection and was considered one of the original conditions for diagnosing AIDS. In Sub-Saharan Africa, KS is one of the most common cancers in menFour clinical variants of this disease have been recognized: classic, endemic, and epidemic subtypes in HIV-positive patients, and the iatrogenic subtype. Type 8 human herpesvirus is the etiologic agent in all of these variantsIn addition to clinical classification, typical KS is histologically categorized into three stages based on disease progression: patch, plaque, and nodular stages. During his initial examination in our service, the patient exhibited all three histologic stages simultaneously. Some parts of the tumor may have regressed due to irregular antiretroviral therapy treatmentWe emphasize the rarity of the case and the importance of identifying certain histologic features, such as promontory signs, that can classify KS stages and predict the optimal treatment and management for the patient.