Staphylococcus aureus (93.0%) and extended-spectrum β-lactamase-producing Escherichia coli (88.7%) in vitro. Moreover, as a potential wound dressing material, the Cy-CBMG hydrogel exhibited excellent biocompatibility and effective antimicrobial ability to promote wound healing in vivo. Overall, this work proposed a practical strategy to synthesize a photosensitizer–excipient compound to enhance the photophysical property and antibacterial efficacy for PDT.As a non-invasive and non-specific therapeutic approach, photodynamic therapy (PDT) has been used to treat antibiotic-resistant bacteria with encouraging efficacy. Inspired by light, the photosensitizers can produce excessive reactive oxygen species (ROS) and, thus, effectively destroy or kill bacteria. Cyanine (Cy), a traditional photosensitizer for PDT, has the advantages of low cytotoxicity and high ROS yield. Yet, the water solubility and photostability for Cy are poor, which substantially limit its antibacterial efficiency and clinical translation. Herein, we combined Cy with carbomer gel (CBMG) to form a photodynamic Cy-CBMG hydrogel. In this system, Cy was evenly dispersed in CBMG, and CBMG significantly improved the water solubility and photostability of Cy via electrostatic interactions. The developed Cy-CBMG gel had less photodegradation under laser irradiation and thus can effectively elevate ROS accumulation in bacteria. The Cy-CBMG compound presented remarkable ROS-induced killing efficacy against methicillin-resistant  Antimicrobial resistance (AMR) is a serious threat for global health and development . SecondaPDT is a photochemistry-based treatment that combines photosensitizers, light, and oxygen. When the photosensitizer is activated by the light of a specific wavelength, local reactive oxygen species (ROS) are elevated to destroy the proteins, nucleic acids, and membranes of the cells, and eventually leads to cell death ,9,10. ClCyanine (Cy), a structural analog of indocyanine green authorized by the Food and Drug Administration (FDA), is an efficient photosensitizer for PDT ,16. WithThe stabilization of the drugs–excipients compound can be enhanced by a number of physical interactions, such as electrostatic interactions, hydrogen bonding, and hydrophobic interactions . While CIn the current study, we synthesized a combined Cy–carbomer hydrogel compound (Cy-CBMG) with enhanced performance of the solubility and photostability. As expected, the developed biocompatible Cy-CBMG can generate excessive ROS under laser irradiation to kill multidrug-resistance bacteria, and then accelerate wound healing, suggesting its possible application in wound dress . β-lactamase-producing Escherichia coli  and methicillin-resistant Staphylococcus aureus  were given courtesy of the Lab of Microbiology at the author’s institute.1,3-Diphenylisobenzofuran (DPBF) and 2,2,6,6-tetramethylpiperidine (TEMP) were purchased from Adamas . Carbomer 940  and triethanolamina  were purchased from Macklin Reagent Co. Ltd. , and Cy  was purchased from Meryer . 2,7-Dichlorofluorescein diacetate (DCFH-DA) dye and CCK-8 kit were purchased form Sigma-Aldrich . A LIVE/DEAD BacLight Bacterial Viability kit was purchased from Thermo Fisher Scientific . Extended-spectrum 2O (ddH2O) and stirred for 30 min at 60 °C in a water bath. Then, 0.25 mL triethanolamine was used to adjust the final pH to 6.5–7.5. Then, Cy in powder form was added in CBMG to prepare 0.03, 0.3, 1, 3 mM Cy-CBMG; the mixture was stirred for 30 min and placed at 37 °C for 24 h. Meanwhile, CBMG was also placed at 37 °C for 24 h. The prepared CBMG and Cy-CBMG were rinsed with ddH2O 3 times and stored in sealed glass tube at room temperature.CBMG was prepared according to a previous report . In brieThe micromorphology of CBMG (0.5 wt%) and Cy-CBMG  were characterized by scanning electron microscope  at 10.0 kV accelerating voltage. Briefly, the prepared hydrogels were pre-frozen at −80 °C overnight, then dry-frozen for 48 h. After that, the dry-frozen hydrogels were sputter-coated with gold under vacuum for 10 min before SEM observation.0) were stored in sealed glass tube with 10 mL PBS at 37 °C. At setting time points, the swollen hydrogels were retrieved, blotted dry with a paper towel and weighed (WS), then freeze-dried again. The swell ratio was calculated from the following equation:The swell ratio of CBMG (0.5 wt%) and Cy-CBMG  was measured for 70 h in the PBS. In brief, the freeze-dried CBMG and Cy-CBMG  was immersed in 20 mL PBS in glass tube. The tubes were sealed and maintain in incubator at 37 °C. At setting time points , 200 μL of supernatant was withdrawn for HPLC-MS/MS, followed by replenished with equivalent fresh PBS.UV-vis absorption spectra were measured with a UV-vis spectrometer . Then, 100 μL of aqueous solution containing 0.3 mM Cy was transferred to a 1 cm quartz cuvette and the absorption spectra was recorded at the wavelength of 200–800 nm.For general appearance and solubility assessment of CBMG, Cy in water (3 mM) and Cy in CBMG (3 mM) were photographed, respectively. Then, Cy in water and Cy in CBMG were centrifuged, and the top layer of samples was transferred to a 1 cm quartz cuvette to acquire the UV-Vis measurements (wavelength range: 200–800 nm).2). At setting times , the UV-Vis measurements of the compound samples were acquired at the wavelength of 200–800 nm.For photostability evaluation, 100 μL Cy in water (0.3 mM) and 100 μL Cy in CBMG (0.3 mM) were transferred to a quartz cuvette, respectively. The samples were irradiated with a 600 nm laser  was detected with an EPR spectrometer , using TEMP as a spin-trapping probe. Typically, 1 μL TEMP was mixed with 100 μL aqueous solution containing 0.3 mM Cy. The solution was irradiated with a 600 nm laser (100 mW/cm2) for 15 min and then immediately transferred to the spectrometer for signal acquisition.The production of 5 cells/well in 24-well-plate for 24 h. After washing with PBS for 3 times, cells were harvested and immobilized with 4% paraformaldehyde for 15 min, infiltrated with 0.1% Triton X-1000 for 10 min, and then sealed with 0.3% bovine serum albumin for 30 min. Cells were incubated in Alexa Phalloidin solution (30 μL/mL of PBS-T) for 30 min for actin cytoskeleton staining, and immersed in DAPI (1:1000 v/v) for 10 min for nucleus staining. After incubation, the cells were washed with PBS 3 times and studied under a confocal laser scanning microscope .To evaluate the biocompatibility of compound samples, human umbilical vein endothelial cells (HUVECs) were cultured with PBS, carbomer (0.5 wt%), Cy (0.3 mM), Cy-CBMG (0.3 mM) at a concentration of 1 × 104 cells/well was cultured with PBS (control), CBMG , Cy , Cy-CBMG  for 24 h, respectively. Cells were then washed and stained with 10% CCK-8 solution for 4 h. The relative cell density was measured using a microplate reader at an absorbance value of 450 nm.CCK-8 kits were used to investigate cell viability for samples at different concentrations of. In brief, HUVECs at a concentration of 1 × 10+) and ESBL E. coli (G−) were employed in experiments. Bacterial cells were cultured in trypticase soy broth (TSB) medium overnight and harvested at the exponential growth phase. The bacteria concentrations were adjusted to 1 × 108 CFUs/mL after centrifuged and re-suspended with fresh PBS.MRSA (GE. coli suspensions (1 × 106 CFUs/mL) and then irradiated with laser  for 30 min. The control groups did not undergo laser irradiation. The antibacterial activity was assessed using the spread-plate method, ROS analysis, scanning electron microscope, and live/dead staining.PBS, Cy , CBMG (0.5 wt%), and Cy-CBMG  were transferred into 1 mL MRSA or ESBL The spread-plate method was used to count the viable bacteria after various treatments. In brief, after being diluted serially, 100 μL bacterial suspension was spread on a sheep blood agar plate and cultured overnight. The number of bacterial colonies was counted for each sample. Bacterial viability was calculated using the following equation: Bacterial viability (%) = E/C × 100%, where C is CFUs in PBS (laser-) group and E represents CFUs in the experiment group.Using 2,7-dichlorofluorescein diacetate (DCFH-DA) dye, ROS analysis was performed to measure the intracellular ROS levels in bacteria after various treatments. Specifically, the as-prepared MRSA were incubated with 10 μM DCFH-DA at a dark room for 30 min. After rinsing twice, the luminescence intensity was detected by a microplate reader (488/525 nm). Then, the stained bacteria were imaged with a confocal laser scanning microscope.E. coli were determined with a SEM. As-prepared bacteria were washed twice with PBS and fixed with 2.5% glutaraldehyde at 4 °C for 4 h. After that, the serially dehydration was performed in ethylalcohol–water hybrid solution  for 10 min, respectively. Then, bacteria were freeze-dried and sputter-coated with gold before SEM  observation.The morphology changes of MRSA and ESBL A LIVE/DEAD BacLight Bacterial Viability Kit was used to examine bacterial viability after treatments. According to the manufacturer’s protocol, STYO-9 and propidium iodide (PI) dyes were used to stain live and dead cells with green and red luminescence, respectively. In brief, the treated bacteria were harvested and stained with dyes  for 20 min in a dark room. The collected bacteria were then rinsed with saline for twice, and then confocal microscope  observations were performed.7 CFUs/cm2) was then smeared in the wound.A mouse model of MRSA-infected wound was used to assess antibacterial activity of the synthesized materials. A total of 80 female Balb/c mice , which were fed in an aseptic environment, were used. Mice were anesthetized by intraperitoneal injection with 3% pentobarbital sodium. The dorsal hair was shaved and sterilized to create a 6 mm × 6 mm full-thickness skin wound using a surgical blade. The MRSA suspension was grown overnight and then collected after rinsing with fresh PBS. The MRSA suspension (1 − 2 × 102) for 30 min (once a day for 2 days). Mice in the other 4 groups were treated with the abovementioned medicines but did not undergo laser irradiation. The effectiveness of the various treatments was evaluated at setting times.Two days later, the mice were randomly divided into 8 groups (10 mice/group) based on laser irradiation exposure and treatments. Before treatment, the wound was photographed, and the time was defined as day 0. For 4 groups, mice were given the following treatments at day 0 and day 1: PBS, Cy (0.3 mM), CBMG (0.5 wt%), or Cy-CBMG (0.3 mM), and further received 600 nm laser irradiation  to assess the healing process. On day 12, all mice were executed using overdose anesthesia, and the wound tissues were collected for Masson trichrome staining. Meanwhile, vital organs  were harvested for HE staining and histological evaluation.t-tests were used for data analysis, as appropriate. p < 0.05 was considered statistically significant.Data were reported as mean ± standard deviation (SD). One-way or two-way analysis of variance (ANOVA) and In this work, we combined carbomer with Cy to form Cy-CBMG hydrogels via electrostatic interactions. Cy-CBMG hydrogels were found to have increased solubility, elevated photostability, and enhanced photo-activated antibacterial activity both in vitro and in vivo, as compared with Cy in water.The micromorphology of CBMG and Cy-CBMG was characterized by SEM. As shown on 1O2), a typical ROS, and demonstrate a decreased absorption intensity at 410 nm [1O2 was examined using DPBF as the probe molecule in water. Cy was irradiated by a 600 nm laser (100 mW/cm2). As the time of light irradiation increased, the absorption intensity of DPBF around 410 nm gradually decreased (1O2 was produced by Cy under laser irradiation (600 nm), with TEMP as the 1O2 trapping agent. As shown in 1O2 was produced by Cy.The capability of producing ROS was determined for Cy with DPBF absorbance and EPR spectroscopy. DPBF can react with singlet oxygen  and ESBL E. coli (Gram−) were used as model bacterial strains. Using the spread-plate method, the cell viability of MRSA and ESBL E. coli after various treatments was determined by assessing colony-forming units (CFUs) on each plate. As shown in E. coli. However, photo-activated Cy and Cy-CBMG exhibited dose-dependent antibacterial activities for MRSA and ESBL E. coli. Taking the biosafety and bactericidal effectiveness of Cy into account, the feeding concentration at 0.3 mM was selected for further evaluation. In this system, photo-activated Cy-CBMG exhibited excellent antibacterial efficiency (93.0%) towards MRSA, which far outweighed Cy dissolved in water (80.7%) and other groups . For other groups, the wound healing rate was lower than 30% at day 4 a,b. In aWhile the wound closure rates in Cy-CBMG/laser group and Cy/laser group were nearly 100% and 87.4% in day 12, respectively, poor wound healing rates were observed in other groups (<75%). Collagen formation is a hallmark of wound regeneration and skin reconstruction . As showIn addition, HE staining of heart, liver, spleen, lung and kidney indicated that no obvious tissue damage was observed in mice in all groups , suggestE. coli. Moreover, as a wound dressing material, the Cy-CBMG hydrogel exhibited excellent biocompatibility and effective antimicrobial capability to accelerate wound healing (wound closure rate was close to 100% after 12-day treatment) in vivo. Results demonstrate that the Cy-CBMG hydrogel is a promising alternative material for antibiotic dressing for infected wounds.The novel compound Cy-CBMG hydrogel developed in the current study, as formed upon electrostatic interactions between Cy and carbomer, had improved solubility and photostability. The laser triggered Cy-CBMG effectively yielded ROS and efficiently suppressed AMR bacterial infection in vitro, with antibacterial efficiency of 93.0% for MRSA and 88.7% for ESBL


Myeloid-derived suppressor cells (MDSCs) are a group of immature and heterogeneous myeloid cells with immunosuppressive functions. MDSCs play important roles in the pathogenesis of cancer, chronic inflammatory diseases, and many autoimmune disorders. The accumulation and activation of MDSCs can be regulated by tumor necrosis factor α (TNF-α). In this review, we summarize the roles played by TNF-α in the recruitment, immunosuppressive functions, and chemotaxis of MDSCs, and discuss the potential therapeutic effects of TNF-α upon these cells in tumor growth and some inflammatory disorders. Inflammation is a process that alerts an organism’s immune system to invasion by pathogens, injuries to tissues, and other attacks. It is also an essential process in the elimination of pathogens and the repair of damaged tissues. However, organisms can be injured by uncontrolled inflammation or constant low-grade inflammation. A balance of inflammation and immune system processes is important, both to maintain normal physiological functions and to promote recovery from disorders .Tumor necrosis factor alpha (TNF-α), a key cytokine linking inflammation and the immune system, can be produced by a variety of cell types throughout living organisms . Anti-TNTNF-α exists in two forms: transmembrane TNF-α (tmTNF-α) and soluble TNF-α (sTNF-α). tmTNF-α is expressed in the form of type II transmembrane peptides composed of 233 amino acids (26 kDa) on the surfaces of activated macrophages, lymphocytes, etc. It is then processed by the TNF-α-converting enzyme and converted to sTNF-α, which contains 157 amino acids (17 kDa). Both forms of TNF-α can be activated through forming trimers [TNF-α exerts effects by binding to its receptors: TNFR1, and TNFR2 . TNFR1 iIn summary, TNF-α can be seen as a double-edged sword. On the one hand, it is involved in maintaining the homeostasis of the immune system and the defenses of the host. On the other hand, TNF-α participates in the pathological processes of malignancies and autoimmune diseases ,18. ReceImmune responses mediated by immune cells derived from bone marrow hematopoietic stem cells have gradually evolved to be the main mechanisms by which a host organism resists infections . HoweverClassical activation of immune cells occurs when the body responds to a strong signal stimulated by a pathogen, such as an acute infection or trauma. At this point, neutrophils and monocytes are rapidly mobilized, phagocytosis is enhanced, major histocompatibility complex class II (MHCII) and co-stimulatory molecules are upregulated, and anti-inflammatory factors such as TNF-α are produced. However, in some chronic diseases, such as chronic infection and cancer, the signals stimulated by the pathogen are weak and of long duration. In such cases, inflammatory factors and endoplasmic reticulum stress are activated. Neutrophils and monocytes now exhibit heterogeneous and immature phenotypes. They exhibit weaker phagocytic activity, and produce more Arg1, ROS, and NO. These pathologically activated cells are known as MDSCs and have specific cell surface markers CD11b and GR1 . CurrentProlonged exposure to TNF-α can suppress the immune systems of animals . In addiThe effects of TNF-α may be crucial for the accumulation of MDSCs in many pathological processes, including tumor growth and chronic inflammation. For example, the TNF signaling promotes the accumulation of MDSCs in the chronic inflammation and carcinogenesis induced by 3-methylcholanthrene (MCA) . HoweverThe authors  found thAs an important kind of immunosuppressive cells, MDSCs exert considerable therapeutic effects in cases of autoimmune disease, organ transplantation, and cardiovascular disease . For thiMDSCs can be induced from PBMCs with the participation of sTNF-α. Hsu et al. found that the combined administration of sTNF-α, GM-CSF and LPS induced MDSCs with an immunosuppressive function from PBMCs . SeledtsMDSCs can also differentiate from bone marrow cells. The authors  reportedIn addition, MDSCs can differentiate from bone marrow cells. It has been reported that PMN-MDSCs can be induced from CD33+ bone marrow by CD4+ T cells, which depend on direct cell-to-cell contact and tmTNF-α signal transduction . Taken tTNF signaling can inhibit the apoptosis of MDSCs in a variety of pathological models, by promoting the accumulation of MDSCs. Qin et al. found spontaneous suppression in the growth of transplanted tumors in Tnfr−/− mice . Zhao etEstrogen also plays important roles in the anti-apoptotic effects mediated by TNF signaling in MDSCs. Dong et al. found that the proportion of peripheral MDSCs is higher in women than men with systemic lupus erythematosus (SLE). The authors  found thIn addition to tmTNF-α/TNFR2 signals, the signaling pathways mediated by sTNF can also alleviate apoptosis in MDSCs. By inducing the activation of NF-κB, upregulating the expression of cFLIP, and inhibiting the expression of caspase-8, sTNF protects MDSCs from apoptosis and prolongs their survival . HoweverThese findings indicate that the apoptosis of MDSCs can be profoundly influenced by TNF-α signaling, and this is likely due to a combination of factors. The ability of MDSCs to inhibit T cell proliferation is well known. Deng et al. studied the interaction between T cells and MDSCs and found that CD8+ T cells induce the apoptosis of MDSCs in a TNF-dependent manner. For this reason, a combined treatment of high-dose ionizing radiation and anti-PD-L1 can downregulate the accumulation of MDSCs by potentiating the activation of CD8+ T cells, which promotes tumor regression to a certain extent .We can now summarize the mechanisms by which TNF signaling pathways facilitate the accumulation of MDSCs, through promoting their development, inhibiting their differentiation and apoptosis, contributing to their induction, etc. Most of the aforementioned mechanisms are mediated by tmTNF-TNFR2. This may be because expression of TNFR2 is much higher than that of TNFR1 in MDSCs. sTNF also plays important roles in the accumulation of MDSCs. During the early stages of tumorigenesis, MDSCs exhibit low levels of expansion. However, MDSC expansion greatly increases with advanced tumor progression. This phenomenon may be explained by the fact that MDSCs are regulated by various kinds of cytokines at different stages of the carcinogenesis process. Specifically, IL-1α may regulate the growth of MDSCs during the initiation of carcinogenesis, while VEGF and GM-CSF promote the expansion of MDSCs after tumor formation ,45. HoweThe interactions of inflammation and its regulatory pathways contribute to an effective immune response that eradicates pathogens while avoiding excessive damage to host cells. MDSCs are a kind of immunosuppressive cells. They help to maintain the immune homeostasis of the body by inhibiting excessive immune response . SystemiImpaired T-cell immunity during chronic inflammation is mediated by MDSCs. Once MDSCs are depleted, the immune function of T cells is fully restored . SignaliFeldman et al. found that TNF-α enhanced the immunosuppressive function of MDSCs in a chronic inflammation model using heat-inactivated pathogens. They also found downregulation of the T-cell antigen receptor ζ chain, dysfunction of T cells, and the appearance of NK cells in vivo . In addiChavez-Gala et al. constructed a mouse model with overexpression of tmTNF. Two subtypes of MDSCs (M-MDSCs and G-MDSCs) were isolated from the tmTNF KI mice and cocultured with TNFR1 or TNFR2 knockout CD4+ T cells, respectively. Compared with the wild-type (WT) mice, the M-MDSCs and G-MDSCs obtained from tmTNF KI mice promoted the proliferation of TNFR2 knockout CD4+ T cells. However, in the case of TNFR1 knockout CD4+ T cells, two subtypes of MDSCs from tmTNF KI mice continued to inhibit proliferation and exhibited a stronger inhibitory effect, compared with those obtained from WT mice. The inhibitory effects of MDSCs on CD4+ T cells, therefore, require the self-expression of tmTNF in MDSCs and TNFR2 in CD4+ T cells .Compared with T cells, there have been fewer studies concerning the effects of MDSCs on B cells. The authors  reportedThe immunosuppressive effects of MDSCs may stem from a physiological regulation process involving negative feedback from T cells. MDSCs and T cells influence each other and, thus, act together to maintain immune homeostasis in organisms. The authors  found thRegarding the immunosuppressive function of MDSCs regulated by TNF signaling pathways, researchers have found that sTNF mediates the acquisition and maintenance of immunosuppressive function in MDSCs, while tmTNF enhances the immunosuppressive function of MDSCs ,32 Figu. In addiIn one study of lung cancer, researchers found that the recruitment of MDSCs decreased in TNF KO mice compared with WT mice, inhibiting tumor growth. Ba et al. conducted studies of Tnfr−/− and WT mice injected subcutaneously with murine hepatic carcinoma cell line H22 cells. They found that TNFR deletion inhibits the accumulation of M-MDSCs and G-MDSCs in spleens and tumor tissue but has no influence on their prevalence in bone marrow. Moreover, the chemotactic ability of TNFR-null MDSCs is weakened because their expression of chemokines such as CXCL12 and CXCR4 decreases obviously .In another study, researchers intervened Tnfr1–/– and Tnfr2–/– mice with sTNF and tmTNF, respectively. They found that tmTNF activates NF-κB and p38 signaling pathways in MDSCs mediated by TNFR2, inducing their expression of CXCR4, and subsequently enhancing the chemotactic ability of MDSCs . In tumoIn this review, we have highlighted the key roles played by TNF-α and TNFR in the accumulation, immunosuppressive function, and chemotaxis of MDSCs. We have analyzed their importance for the control of inflammation and tumor progression, and discussed the potential targeting effect of these signaling pathways, thus providing new ideas for the immunotherapeutic treatment of health disorders and disease.TNF-α is a potent pro-inflammatory factor and exerts pleiotropic effects in a variety of cell types. TNF-α plays critical roles in some tumors or chronic inflammatory diseases by influencing MDSCs, which are an important population of immunosuppressive cells. For this reason, researchers are now focusing on regulating TNF-α or its signaling pathways in MDSCs with the objective of developing new disease treatments. Several recent studies are summarized in The data presented in this table are all from preclinical studies. In this review, we summarized the pharmacological and biological agents targeting TNF signaling, which have promising therapeutic perspectives by regulating the recruitment and immunosuppressive function of MDSCs. Although TNF inhibits the apoptosis of MDSCs, it is not the most efficient member of the family to trigger apoptosis. Signaling through Fas, TRAIL-R1 and TRAIL-R2 has been well characterized . EngagemFurthermore, there are also some pharmacological agents that target TNF-α, such as adalimumab, golimumab and trastuzumab, some biological agents that target sTNF/TNFR1, such as TNFR1-selective antagonist R1antTNF and TNFR1-specific antibody DMS5540, and some agents that target tmTNF/TNFR2, such as TNFR2-specific neutralizing antibody, and CD3-specific antibody hOKT3γ1(Ala-Ala) . The effTNF-α can be seen as a double-edged sword. On the one hand, TNF-α can maintain the host defense function and the immune homeostasis. On the other hand, it is involved in the pathological process of malignant diseases, autoimmune diseases, and inflammatory disorders ,18. AlthTNF-α is widely expressed throughout the body and mediate wide-ranging physiological roles. Therefore, cancer risk and other serious side effects should be monitored when using TNF-α or its inhibitors for treatment ,60. In tIn conclusion, our review summarized the regulation and mechanisms of TNF signaling on the recruitment, immunosuppressive function, and chemotaxis of MDSCs in certain tumors, chronic inflammatory diseases, and some autoimmune disorders. Regulating TNF-α signaling pathways in MDSCs may be another therapeutic strategy, which provides new ideas and directions for future studies.


Gamification in medical education has gained popularity over the past several years. We describe a virtual escape box in emergency medicine clerkship didactics to teach chest pain and abdominal pain and compare this instructional method to a traditional flipped classroom format.A virtual escape box was designed at our institution and incorporated into the mandatory two-week emergency medicine clerkship. The game consisted of a PDF with four cases containing puzzles to unlock a final clue. Likert scale surveys were administered to assess participants’ perceptions of the escape box format; of clerkship didactics as a whole; and of the clerkship overall. These responses were compared to the prior year’s evaluations on flipped classroom didactics and clerkship.p = 0.0491) for the category of “lecturer explaining content clearly and at the proper level of complexity.” For the category of “lecturer provided effective instructional materials,” the escape box showed higher scores (3.6 ± 0.63) compared to flipped classroom for chest pain (3.4 ± 0.77) and abdominal pain (3.1 ± 0.80) (p < 0.001).One hundred thirty-four learners participated in the escape box and completed the survey. Eighty-six percent strongly agreed with feeling more engaged with the escape box, 84% strongly agreed with learning something new, 81% strongly agreed with finding the escape box to be satisfying, 78% strongly agreed with being able to apply knowledge gained, and 74% strongly agreed with wanting more escape boxes incorporated into medical education. The escape box showed a higher average score (3.6 ± 0.63) compared to chest pain (3.5 ± 0.67) and abdominal pain (3.2 ± 0.77) flipped classroom sessions (Escape boxes are adaptable to a virtual format and can teach abstract concepts such as teamwork and communication in addition to traditional lecture content. Ratings of didactics were higher for the escape box compared to the flipped classroom, while ratings of overall clerkship experience were not found to change significantly.The online version contains supplementary material available at 10.1186/s12909-022-03585-3. Gamification is the incorporation of game elements into a non-game setting. In medical education, gamifying traditional lecture-based material has gained popularity as alternative methods of instruction. The rise of gamification in the medical education setting coincides with the rise of a new generation of medical learners. Guckian et al  coin theAn escape room is a game style that provides an immersive experience for learners. Often accompanied by a storyline, escape rooms incorporate a series of puzzles (puzzle hunt) and tasks, requiring teams to complete the tasks within a time constraint to escape the room or unlock a box . Escape With the transition to distance learning, the need for virtual instruction became paramount to prevent discontinuity in medical education. Here we describe a virtual escape box for an emergency medicine (EM) clerkship for third- and fourth-year medical students to teach cardiac and abdominal pathology and management. We sought to determine learners’ attitudes towards gamification in this context – including utility, engagement, satisfaction, and desire to have more game elements in medical education – and compare the virtual escape box to a traditional flipped classroom format in the prior academic year.The study was reviewed by the Institutional Review Board and determined to be self-exempt based on institution protocol. The escape box materials were designed for compatibility with a virtual, online learning platform through a 1-h video conferencing call with a lead lecturer.The objective of the escape box was to solve multiple puzzles within the four cases in order to decode a four-letter final clue, all within 30 min. The topics covered were chest pain and abdominal pain. Learning objectives from the case-based session were mapped to the puzzle to ensure that objectives would be met through the game process. Students were presented with learning objectives prior to starting the game when downloading the case materials. Students electronically received a PDF document containing cards for four patients. Learners were required to work together in small groups in Zoom breakout rooms to uncover passwords that were used to open QR codes to end each case. The four cases involved puzzle elements consisting of: prioritizing interventions in the correct order to reveal a permutation of letters, matching anatomic areas of an electrocardiogram to generate a three-number code, matching diagnoses to their respective radiologic findings, and summing values assigned to associated symptoms to generate another code. QR codes were used to reveal four single-letter clues that opened the escape box once all four letters were collected. At the end of the game, students left the breakout rooms and joined together in the main Zoom room. The attending lecturer led a 20-min slide-based debrief reviewing all game puzzles and relevant learning objectives related to those puzzles.Our survey study compared the replacement of the traditional flipped classroom format with the escape box game. In previous years, students had two 1-h case-based flipped classroom learning sessions that took place in-person regarding topics in emergency medical care for patients presenting with chest pain and abdominal pain. In the flipped classroom year, learning objectives were also clearly stated on the webpage where students downloaded materials for the session. The flipped classroom format requires students to complete readings and/or watch videos on their own, prior to attending an in-person session. In an effort to meet the needs of a new generation of learners as well as comply with social distancing requirements and distance learning during the COVID-19 pandemic, a gamified version of these didactics was created as a flipped classroom substitute. Additionally, in the escape box format, chest pain and abdominal pain chief complaints were combined into one 1-h session.This survey study took place at an academic hospital and level 1 trauma center. Our institution requires all medical students to complete a two-week clerkship in emergency medicine as a graduation requirement. Students take the two-week rotation throughout the academic year with a maximum of 4 students per rotation. Per institution policy, the clerkship provides four hours of medical student didactics to learners on the rotation over the two weeks. Our intervention takes place in the first week’s learning session.Three surveys were used to evaluate our escape box intervention. Students in the 2020–2021 academic year completed all three surveys. Students in the 2019–2020 academic year completed only the post-didactic and post-clerkship surveys. The post-escape box survey was completed only by students in the 2020–2021 academic year to assess students’ perceptions about the game immediately after the session was completed and in isolation from the other didactic activities offered during the two-week clerkship. This post-escape box survey was used to evaluate solely the gamified didactics, whereas the post-didactic survey and post-clerkship survey evaluate clerkship didactics as a whole and is implemented by the school of medicine as a requirement for clerkship evaluation. These post-didactic and post-clerkship surveys were already in place to evaluate didactics overall and the clerkship once completed.Students completed an anonymous, voluntary, online survey immediately after the escape box session. Questions were graded on a Likert scale of strongly disagree to strongly agree, with numeric scoring from 0 (strongly disagree) to 4 (strongly agree). Questions pertained specifically to the escape box didactics format regarding: level of engagement; ability to apply knowledge from the activity in a clinical setting; satisfaction of the escape box educational experience; learning something new through the escape box; and desire for more escape boxes to be incorporated into medical school training.At the end of the clerkship, once all didactics were completed, students received an automated email to complete an anonymous, voluntary, online survey assessing the entire didactic curriculum (including the escape box experience and case-based learning). Questions in this survey were graded on various Likert Scales from 1–4 that asked students to evaluate facilitator teaching, instructional materials, teaching effectiveness, satisfaction and relevance of the didactic session as a whole.At the conclusion of the two-week clerkship, students were encouraged to complete an online, post-clerkship survey administered by the institution. While students were asked to evaluate all aspects of the clerkship, students were specifically asked to rate  the quality of seminars, teaching and clerkship experience.τ) and p-value. Type I error was set to 0.05. SPSS Statistics version 26  was used for the analysis.The sample size was 134 for students who took the post-escape box survey. For the post-didactics survey, 57 students completed the survey for the escape box; 64 students completed the chest pain flipped classroom survey; and 49 students completed the abdominal pain flipped classroom survey. For the post-clerkship survey, 89 students completed the survey in 2020–2021 and 87 students completed the survey in 2019–2020. Distribution of participants’ answers are reported as absolute (N) and relative frequencies (%). Scores are reported as mean ± standard deviation (SD) and were compared between the three study groups by using analysis of variance (ANOVA). Correlation between the different questions is reported as Kendall's tau correlation coefficient (rn = 115) strongly agreed with feeling more engaged with the escape box design, 84% (n = 112) strongly agreed with learning something new, 81% (n = 109) strongly agreed with finding the escape box to be a satisfying experience, 78% (n = 104) strongly agreed with being able to apply knowledge gained from the experience, and 74% (n = 99) strongly agreed with wanting to incorporate more escape boxes in medical education . The highest correlation was seen with finding escape boxes to be a satisfying activity  . For the category of “lecturer provided effective instructional materials,” the escape box also showed higher scores (3.6 ± 0.63) compared to chest pain (3.4 ± 0.77) and abdominal pain (3.1 ± 0.80) (p < 0.001).We next compared differences between lecture evaluations from the 2019–2020 academic year, during which a flipped classroom model was used to teach chest pain and abdominal pain in separate sessions, and the evaluations from the 2020–2021 year, during which the flipped classrooms were replaced with a single escape box session incorporating both topics. The same group of lecturers led the sessions in both the 2019–2020 and 2020–2021 academic year. Direct comparison of a single lecturer between academic years was not feasible due to the year-round nature of the clerkship and availability of the lecturers. Fifty-seven students completed the post-didactics survey for the escape box, 64 students completed the survey for the chest pain flipped classroom, and 49 students completed the survey for the abdominal pain flipped classroom. The mean scores and standard deviations are shown in Table The escape box format for teaching chest pain and abdominal pain were predominantly well-received, with the vast majority of learners strongly agreeing with survey questions on engagement, learning, satisfaction, application, and desire for further escape box use. Learners who strongly agreed with incorporating escape boxes in the medical education setting also were most likely to strongly agree with reporting satisfaction as a learner. When stratified by medical school training level , fourth-year students tended to rate knowledge application, satisfaction, and desire for more escape boxes more highly than third-year students, though not to a significant degree.In comparing students’ perceptions of didactics quality between escape box and flipped classroom, there were significant increases in rater approval in the escape box group for explaining content clearly and at the appropriate level of complexity, as well as in the category of providing effective lecture materials. While the escape box cohort learned abdominal pain and chest pain during a combined 1-h session compared to the flipped classroom cohort, which dedicated an hour to each topic separately, learners had more favorable perceptions of the escape box format in rating clear explanation of content. A possible explanation for this finding could be higher learner engagement in a game setting and more interaction with the facilitator in the discussion portion. There were also clear tasks for each puzzle within the escape box session. On the other hand, in the flipped classroom model, students were presented with a case stem in the form of a paragraph of text and open-ended questions were posed to the group. Although less time was spent in the escape box didactics sessions compared to flipped classroom, learners appear to be more receptive to the material when presented in a game style.The ratings for the emergency medicine clerkship overall did not differ between academic years to a significant degree in categories relating to quality of conferences, experience, and teaching in the clerkship.Tomaselli et al  reportedIn a gamification study by Gerard et al , medicalDeci and Ryan  first deUse of game elements in medical education can also serve as a means to teach processes and more abstract lessons of communication and teamwork. Guckian et al  examinedSince the 2020–2021 academic year was the first year in which the escape box was utilized, further studies would be necessary to increase sample size. Additionally, pre- and post-testing could be utilized to assess learners’ knowledge acquisition from the activity. There is also a temporal element that is difficult to control for, in that students participate in the emergency medicine rotation at different times of the year based on scheduling. As students progress through training, they will naturally acquire more knowledge, which can artificially inflate scores later in the year. Similarly, the data included in our study contained evaluations from third- and fourth-year students, and the differing training levels may be confounding variables. Additionally, lecturer availability varies throughout the year, so the variety of lecturers may also be a confounding variable in having different teaching styles; however, to mitigate this effect, the group of lecturers who delivered content for the flipped classroom and escape box was consistent between the two years.The escape box format is well-received by learners and shows improved ratings in appropriate level of complexity and effective learning materials when compared to a traditional flipped classroom format. Gamification in medical education can provide a means to teach both concrete topics, such as disease diagnosis and treatment, as well as abstract skills, such as communication and teamwork. Further studies with pre- and post-game testing are needed to assess knowledge acquisition using this format of instruction in EM clerkship didactics.Additional file 1. EM Clerkship Escape Box.Additional file 2.Escape Box Learning Objectives.Additional file 3. Data set.


Toumeyella parvicornis . This species is a soft scale insect native to North America recently introduced in Italy and in France, where it established and spread, causing harmful infestations on stone pine (Pinus pinea L.) plants. Some aspects of its biology, such as the several overlapped generations and the high fecundity, may contribute to make it a successful invasive species when in a suitable environment. This is supported by the observations carried out in Central Italy, where the climate and environmental conditions seem suitable for the species to develop. To prevent further spread across the Mediterranean basin, it would be helpful to identify the most suitable areas by considering bioclimatic variables, as is commonly carried out in case of invasive species. We prepared potential pest distribution maps of European areas by utilizing Species Distribution Models. This information adds further detail to the report recently published by the European Food Safety Authority (EFSA). The areas with the highest suitability for the species are located along the coasts, where most Mediterranean pines occur. This correspondence suggests a high risk of widespread dispersal and provides useful information for implementing management strategies of this damaging pest.Predicting species distribution is a fundamental step for setting up opportune control actions. The suitability of the environment for the establishment of the species is even more important in case of invasive insects, such as Toumeyella parvicornis, is an insect native to the Nearctic region that is able to infest several Pinus species. It can cause weakening, defoliation and, at high infestation levels, tree death. After its first report in Italy in 2015, the pest spread rapidly over the surrounding areas and was reported in France in 2021. Due to the threat that this pest poses to pine trees, the suitability of European Mediterranean basin areas for T. parvicornis at different spatial scales was estimated by constructing species distribution models (SDMs) using bioclimatic variables. Our results showed that several coastal areas of the Mediterranean basin area could be suitable for T. parvicornis. Based on performance assessment, all the SDMs tested provided a good representation of the suitability of European Mediterranean non-native area for T. parvicornis at different spatial scales. In particular, most of the areas with a medium or high level of suitability corresponded to the geographical range of distribution of different Pinus spp. in Europe. Predicting the suitability of European Mediterranean areas for T. parvicornis provides a fundamental tool for early detection and management of the spread of this pest in Europe.The pine tortoise scale,  Toumeyella parvicornis (Cockerell) (Hemiptera: Coccidae), is a sap-sucking insect infesting several species belonging to Pinus genera (Pinaceae)  is the primary host of this pest  and the European and Mediterranean Plant Protection Organization (EPPO) database . At each selected location, T. parvicornis infestations were assessed by visually inspecting the trees for typical symptoms ] as georeferenced raster files . Multicollinearity among the bioclimatic variables was tested at a spatial resolution of 30 arc-sec (∼1 km2) using the Pearson’s correlation coefficients in order to remove the highly correlated variables (R2 > 0.80 or R2 < −0.80) before running the models  [er files . Georefee models . The mule models .T. parvicornis in Italy and Europe: Generalized Linear Model (GLM), Multivariate Adaptive Regression Splines (MARS), Random Forest (RF), and MaxEnt [Four different algorithms were used to estimate the potential distribution of d MaxEnt ,34. The d MaxEnt  in R sofd MaxEnt .The GLM, MARS and RF algorithms need pseudo-absence records in addition to occurrence points. Therefore, 1000 pseudo-absence records, located at least 500 m apart from the occurrence points, were generated. This process was repeated 3 times, thus obtaining three different datasets of pseudo-absences for each algorithm. The default model settings in biomod2 were used for fitting GLM, MARS, and RF algorithms.The MaxEnt algorithm can be applied considering presence-only records instead of presence-absence data , which iParameterization in MaxEnt is necessary as models fitted with default settings often lead to skewed projections if compared to the parametrized models ,40,41,42For all the tested models, 80% of the data was used for calibration, whereas the remaining 20% was used for performance tests, as conventionally applied for SDM application and modelling ,46,47.Model performance was evaluated by considering the Area Under the Curve (AUC), the Receiver Operating Characteristic (ROC), and the True Statistical Skill (TSS). In short, AUC measures the discrimination capacity between presences and absences and ranges from 0 to 1. Values of AUC higher than 0.8 and 0.9 indicate good and excellent performances, respectively ,49,50,51T. parvicornis development. The variables with the greatest contributions to the distribution of T. parvicornis were conventionally divided in “classes of suitability” that ranged from “very low” to “very high” suitability (S), as follows: S < 0.2 = very low, 0.2 ≤ S < 0.4 = low, 0.4 ≤ S < 0.6 = intermediate, 0.6 ≤ S < 0.8 = high, and S ≥ 0.8 = very high.Finally, the distribution probabilities obtained by the models were projected using QGIS software to visually evaluate the suitability level of European countries for T. parvicornis varied with the model involved. The bioclimatic variables influencing the most the distribution of T. parvicornis in Italy were BIO01  and BIO12  for the GLM model, BIO01 and BIO03  for the MARS model, BIO03 and BIO01 for the RF model, and BIO01 and BIO15  for the MaxEnt model  had the best performance compared to the other tested models .Assessment of SDMs performance when determining the areas suitable to T. parvicornis were identified among the tested SDMs. The potential T. parvicornis distribution area was mainly concentrated around the coastal areas of Lazio, Campania, and Toscana regions  and BIO19  variables were the most important variables affecting the distribution of T. parvicornis for the GLM and MARS models , followed by GLM , MaxEnt , and RF , which showed very similar values.T. parvicornis in European Mediterranean area were concentrated in the coastal zone  and nonparametric models  [The recent detection of d spread ,55,56,57.g., RF) .T. parvicornis in Italy, especially in the GLM, MARS, and MaxEnt models. This information is of fundamental importance in the framework of a climate change scenario, as already stated for other pest species infesting agricultural and forest environments, such as Philaenus spumarius L. (Hemiptera: Aphrophoridae) [Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) [Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) [Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), Apodiphus amygdali (Germar) (Hemiptera: Pentatomidae), Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), and Thrips palmi Karny (Thysanoptera: Thripidae).Based on our study, the annual mean temperature played a fundamental role in explaining the projections of habitat suitability for horidae) , Lobesiaricidae) , and Tutchiidae) . Our reschiidae)  and Yan chiidae) , where tT. parvicornis by leading this species to have more generations than in places with colder winters. In fact, this phenomenon has been already observed for other alien species, such as Nezara viridula (Linnaeus) (Hemiptera: Pentatomidae) and Hyphantria cunea (Drury) (Lepidoptera: Erebidae) [T. parvicornis.A secondary variable that is worth mentioning for Italy is isothermality, which was the best explanatory variable according to the RF model. This variable is strictly related to the maritime areas, given that it evaluates the thermal gap between summer and winter temperatures . Areas crebidae) ,65,66,67T. parvicornis activity is higher, may act as a natural control of preimaginal stages . In fact, intense rainfall can kill the young instars and the mobile stages of this insect or carry them away from the host plant [Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae), Quadricalcarifera punctatella (Motschulsky) (Lepidoptera: Notodontidae), and Eriogaster lanestris (Linnaeus) (Lepidoptera: Lasiocampidae) [The European scenario outputs provided more extensive projections than the ones obtained that only considered Italy. In fact, the GLM and MARS models confirmed that temperature was the main explanatory variable at European Mediterranean spatial scale, whereas MaxEnt and RF considered precipitation seasonality and precipitation of the coldest quarter, respectively, as the best ones. Precipitation seasonality is a relevant variable, above all, in the coastal areas featured by rainfall anomalies during the seasons  and wherst plant , as alreampidae) ,69,70. Iampidae) .T. parvicornis occurred as a method to predict the potential distribution of the insect.Our findings are also in line with Bragard et al. , who fouT. parvicornis occurring in Europe is P. pinea. This plant species has a low genetic diversity [Pinus species are present. This aspect has been already observed in intensive monoculture fields where a low genetic diversity of the plants resulted in a higher susceptibility to both biotic and abiotic stresses [The main host species of iversity ,73,74, wstresses ,76,77.T. parvicornis may potentially change the aesthetic of many places if no prompt actions are carried out. Aesthetics is not the only element that may be impaired by the pest activity because T. parvicornis infestations may represent a serious concern where stone pines are cultivated for pinenut production, especially in some widely extended areas of Spain, Portugal, and France. Therefore, the interpretation of SDMs’ projections cannot ignore the host plant distribution. From this point of view, our results are in line with the suggestion provided by the European Food Safety Authority [T. parvicornis has the potential to establish throughout the Europe wherever suitable hosts occur. In fact, the most suitable areas for T. parvicornis in European Mediterranean countries estimated by SDMs overlapped with the distribution of P. pinea and P. pinaster  (Hemiptera: Pseudococcidae), another invasive pest infesting the same host plant [T. parvicornis given the extensive presence of host plants and the high suitability indicated by our results.In this regard, based on the information available on the distribution of the main host plant species in Italy and Mediterranean Europe, we analyzed where the potential spread of al areas , where tal areas ,80. Accost plant ,82. Simist plant  and wherT. parvicornis has not been detected yet but stone and maritime pines are widely diffused [A different scenario concerns Sardinia, where diffused ,85, abovT. parvicornis is an oligophagous species strictly related to a restricted pool of host plants, it has several characteristics of an ideal invasive species. In fact, small size, parthenogenetic reproduction, and high adaptability to new environments are features generally associated with highly invasive pests [T. parvicornis makes the detection of the infested plants, especially adult plants, very difficult and promotes its diffusion by wind. In addition to being small, the Italian and European climate conditions seem to play a fundamental role in its diffusion. For instance, in North America T. parvicornis has one generation per year [Although ve pests . Moreoveper year ,87, wherper year ,6.P. pinea and P. pinaster in Europe and our SDMs outputs, we hypothesize that the Iberian Peninsula may be another highly suitable area to T. parvicornis, given the extensive presence of the host plant [Bursaphelenchus xylophilus Nickle (1981) [Tomicus genus [T. parvicornis could be more aggressive towards plants already affected by other adversities.Considering the distribution of both st plant ,89. Extest plant , where tst plant . Howeverst plant ,92, and e (1981) ,94 or baus genus ,96. EvenT. parvicornis for different regions would be of fundamental importance, above all to construct prompt monitoring and control strategies. An early control strategy would avoid some dangerous conditions, as already observed in the Turks and Caicos Islands, where T. parvicornis dramatically reduced the range of native Pinus caribaea var. bahamensis [Obtaining a complete and detailed potential distribution map of hamensis . Besideshamensis ,98.T. parvicornis, thus helping them to manage properly this injurious pest in Mediterranean European area.In conclusion, we believe that the scenario presented in this work could allow technicians and local authorities to focus on the most susceptible areas to


The literature of neuroimaging meta‐analysis has been thriving for over a decade. A majority of them were coordinate‐based meta‐analyses, particularly the activation likelihood estimation (ALE) approach. A meta‐evaluation of these meta‐analyses was performed to qualitatively evaluate their design and reporting standards. The publications listed from the BrainMap website were screened. Six hundred and three ALE papers published during 2010–2019 were included and analysed. For reporting standards, most of the ALE papers reported their total number of Papers involved and mentioned the inclusion/exclusion criteria on Paper selection. However, most papers did not describe how data redundancy was avoided when multiple related Experiments were reported within one paper. The most prevalent repeated‐measures correction methods were voxel‐level FDR (54.4%) and cluster‐level FWE (33.8%), with the latter quickly replacing the former since 2016. For study characteristics, sample size in terms of number of Papers included per ALE paper and number of Experiments per analysis seemed to be stable over the decade. One‐fifth of the surveyed ALE papers failed to meet the recommendation of having >17 Experiments per analysis. For data sharing, most of them did not provide input and output data. In conclusion, the field has matured well in terms of rising dominance of cluster‐level FWE correction, and slightly improved reporting on elimination of data redundancy and providing input data. The provision of Data and Code availability statements and flow chart of literature screening process, as well as data submission to BrainMap, should be more encouraged. Cluster‐level FWE became the mainstream in recent years for ALE‐type CBMA.More ALE papers should exclude ROI papers and report details on data redundancy filter.They should provide Data/Code availability statements and flow chart of paper screening. Based on the list, papers published from 2010 until 2019 were screened (n = 867). We queried the PubMed database by the searching “activation likelihood estimation,” “coordinate‐based meta‐analysis,” or “ALE meta‐analysis,” and returned with 568 papers. It implied that the list from BrainMap page is reflective of the actual literature without missing many papers. Papers were excluded with the following reasons: (1) no ALE meta‐analysis results (n = 254). These excluded papers mainly dealt with multi‐variate modelling only  instead of ALE; (2) not journal publications ; (3) not retrievable (n = 2) and (4) not written in English (n = 3). Finally, 603 publications reporting ALE meta‐analyses were included for the subsequent evaluation.All publications listed in the BrainMap Publications page .Total number of source papers and a list of all of them (yes/no).Data Availability and Code Availability statements anywhere in the text (yes/no). The provision of these information supports open science and facilitates study replication  and provision of data searching statements  to illustrate the data screening and pruning process (yes/no).Statement of inclusion/exclusion criteria (yes/no).Explicit exclusion of papers reporting ROI results only (yes/no). ROI papers should be excluded as they create bias in spatial convergence . Cluster‐level inference should enable a more optimized result.Choice of most stringent statistical correction . Voxel‐wise FDR correction is nowadays considered unsuitable for ALE or even neuroimaging data due to high false positive rates .Data redundancy filter . Treating multiple dependent datasets as independent may exaggerate the influence of the concerned subjects and bias the results .Dataset availability as supplementary files or links to repository .BrainMap as one of the searched databases for Paper identification (yes/no).All five authors designed a data extraction sheet together. One author (AWKY) extracted data from the 603 publications to be double checked by another author (SBE). Internal data from the BrainMap database was provided by two authors (MR and AU). For each publication, the following data were extracted:For point (15), it is likely that many contributors/authors may code and submit data to the BrainMap database without explicitly mentioning it in their paper. Therefore, we also obtained the internal statistics from the BrainMap team to see how many source papers were coded by internal and external contributors respectively during the study period (2010–2019). This should provide a more balanced account of the contributions by the community to the BrainMap database.2.3Statistical analyses were conducted in SPSS 26.0 . Linear regressions were conducted to evaluate if linear trends existed for the annual number of ALE papers published and journal impact factor. Then, tests were conducted to evaluate three aspects: reporting standards, study characteristics, and data sharing. For reporting standards, binomial logistic regressions were conducted to evaluate if publication year was an influencing factor on the ratio of papers that provided a list of all source papers, Data and Code availability statements, flow chart that illustrated the literature screening process, data searching statements, the inclusion/exclusion criteria, and explicit exclusion of ROI papers. Multinomial logistic regressions were conducted to evaluate if publication year was an influencing factor on the choice of statistical threshold, and strategy to adjust for multiple related Experiments from the same set of subjects.For study characteristics, linear regressions were conducted to evaluate if linear trends existed for the number of Papers (source papers) in each ALE paper, number of ALE meta‐analysis per paper and number of Experiments per analysis. Binomial logistic regression was conducted to evaluate if publication year was an influencing factor on the ratio of papers searching of BrainMap database as one of their data sources.For data sharing, multinomial logistic regressions were conducted to evaluate if there were trends in the contributions to BrainMap database, and data availability (provision of input and output data).t‐tests were conducted to evaluate if these groups differed in terms of mean number of source papers analysed, journal impact factor, number of ALE meta‐analyses, and number of Experiments per analysis. Chi‐squared tests were conducted to evaluate if these groups differed in the ratio of stated inclusion/exclusion criteria, explicit exclusion of ROI papers, data redundancy filter, data availability and statistical threshold.We reasoned that papers that supplied data to BrainMap database (provided “Complete Citation” or brain coordinates not indexed in the database) might welcome others to examine their data and thus their reporting standard and study characteristics could potentially be of higher standard. Hence, papers were then divided into two groups, those with contributions to BrainMap database and those without. Independent sample n = 283) had >28 source papers analysed, whereas less‐powered studies (n = 318) had 28 or fewer source papers analysed. Chi‐squared tests were used to evaluate if the two groups differed in terms of ratio of adjusting for multiple related Experiments, explicit exclusion of ROI papers, and use of cluster‐level FWE. The comparisons were then repeated by splitting the papers into those having a mean of >17 Experiments per analysis (n = 126) against those <17 .Reporting standards between well‐powered and less‐powered papers might also be different, because well‐powered papers could spare/exclude unsuitable data and adopt a higher reporting standard. Therefore, papers were first divided into two groups by median splitting their number of source papers analysed. Better‐powered studies . The number of ALE papers published per year rose sharply during the first half of the decade . The use of flow chart and Data availability statement were scarce , whereas Code availability statement was common (73.8%). Their usages showed an increasing trend , a list of all source papers (95.4%), and inclusion/exclusion criteria on Paper selection (96.2%). Meanwhile, only 68.0% of the papers explicitly excluded ROI papers, with the annual ratio always exceeding 65% since 2011 . This change corresponds to  and cluster‐level FWE 33.8%). Voxel‐level FDR has been the predominant choice in the early 2010 s accounting for >80% of papers per year . For those paper that mentioned this, selecting the most representative Experiment was more common than merging multiple Experiments into one Figure . The forMost papers did not explicitly mention that they double‐checked with the original sources about the accuracy of the coordinates to be entered into the meta‐analysis (90.7%).3.3p = .360, Figure p = .031). Each ALE analysis had an average of 42.0 Experiments, which showed no significant trend (p = .457). About one‐third  of the papers had an average of >17 Experiments per analysis as recommended.Most ALE papers reported the total number of Papers involved (99.7%), with no significant trend over the survey period (p < .001). They were also more likely to apply data redundancy filter . The two groups did not differ in terms of explicit exclusion of ROI papers . If we only considered papers reporting number of Experiments per analysis, then better‐powered studies  were more likely to use cluster‐level FWE than less‐powered studies . They did not differ in terms of adjusting for multiple related Experiments  or explicit exclusion of ROI papers .Better‐powered studies  were more likely to use cluster‐level FWE than less‐powered studies , but there was an overall increasing trend in its searching . Over the year, the ratio of ALE papers providing neither input data file (in .txt or any other text format for GingerALE) nor output file always exceeded 90%, except in 2019 the ratio dropped to 80.7% Figure . The ratMost papers did not explicitly mention that they contributed to the BrainMap database 91.5%). In other words, they did not provide “Complete Citation” or use Scribe. The ratio of having contributions never exceeded 25% in a single year Figure , and the%. In othp < .001). They did not significantly differ in terms of journal impact factor, number of ALE meta‐analyses, number of Experiments per analysis, ratio of stated inclusion/exclusion criteria, explicit exclusion of ROI papers, data redundancy filter, data availability and statistical threshold (p > .05).Papers with contributions to BrainMap database had a significantly lower mean number of source papers analysed  than their counterparts . Conduct exhaustive search second.State CBMA standards compliance of input data: whole‐brain, voxel‐wise analyses; comprehensive coordinate reporting; exclusion of ROI studies.State CBMA analysis choices: correction for multiple comparisons; data‐redundancy filter method, and so on.BrainMap.org) all papers not already in BrainMap. State this in Data Availability.Code (Scribe) and submit  output file.BrainMap.org/pubs after acceptance, to create a “complete citation.”Contribute your paper and supplementary materials to Contribute output file (.nii) to ANIMA database.4.1Cluster‐level FWE replaced voxel‐level FDR in 2016 and since has been the most popular inference method. It could be related to the publication of the seminal paper by Eickhoff et al. in that year, which through computation models strongly advocated the use of the former as the most appropriate method and the latter as a method to “be avoided”  and number of foci. The variability/flexibility in the analytic pipeline of neuroimaging data  has been constantly argued as a problem leading to reproducibility issues , these studies only accounted for 1/8 of all surveyed ALE papers. Treating dependent Experiments as independent might pose a potential bias from within‐group effects, which might significantly influence the final meta‐analytic outcome .On the bright side, the neuroimaging community actually largely supported and contributed to the content of the BrainMap project as external contributors coded 61% of the 4826 source papers added to the BrainMap database. It is understandable that the authors seldom mentioned their contributions explicitly in their ALE papers, as the contribution is not directly related to the research methods and results, and the contribution can be done post publication. Therefore, we urge researchers to code all papers with Scribe, submit them to BrainMap, and state this in the Data Availability paragraph to inform readers as a formal documentation. BrainMap workspace and GingerALE input file should be provided as supplementary materials and all these files should be provided to 4.3Many journals nowadays request Data and Code Availability Statements as components separated from the main text, for instance, journals from Public Library of Science (PLOS), Springer Nature, BMC, and member journals of International Committee of Medical Journal Editors (ICMJE)  sector. The present paper addresses solely TA‐sector data. Reports of ALE application to voxel‐based morphometric (VBM) data, however, are growing in number. VBM studies report groupwise, case–control contrasts comparing a disorder  to a control group. VBM data are shared in the BrainMap VBM Sector  hosted by the Texas Advanced Computing Center (www.tacc.utexas.edu). The BrainMap Portal hosts all three BrainMap data sectors  and the BrainMap software suite in a high‐performance computing environment. The portal also provides containerized meta‐connectomic applications  and a venue for sharing workspaces, algorithms and pipelines. The intention is to promote scientific transparency, rigor and reproducibility by openly sharing access to source data, algorithms and results for CBMA.Multivariate CBMA is another area of steady growth in the field, for which reporting standards are not yet well described and which was not addressed in the above‐described analyses. Multivariate CBMA is used to discover brain network properties, both structural and functional, that is, meta‐connectomics. Although meta‐connectomic methods use the same input data (standardized coordinates from published reports), the analyses applied be ALE‐based such as meta‐analytic connectivity modelling .Data sharing: External contributors coded 61% of the 4826 source papers added into the BrainMap database during the period of 2010–2019. This ratio could only be demonstrated using data obtained by the BrainMap team but not from the content of the published papers, because the authors seldom mentioned their contributions to the BrainMap database explicitly.See Box Through the survey of 603 ALE papers published in the 2010s, the following conclusions could be made:The authors have no relevant financial or non‐financial interests to disclose.This work did not involve human or animal subjects and hence ethical approval was not applicable.


TNF-A, FCGR2A, TNFAIP3, TNFR1/TNFR1A/TNFRSF1A, TRAIL-R1/TNFRSF10A, FCGR3A have been reported to correlate with TNF-i response at various levels of statistical significance in patients with PsA. However, results were often from heterogenous and underpowered cohorts and none are currently implemented into clinical practice. External validation of genetic biomarkers in large, well-documented cohorts is required, and assessment of the predictive value of combining multiple genetic biomarkers with clinical measures is essential to clinically embed pharmacogenomics into PsA drug management.Psoriatic arthritis (PsA) is a heterogeneous chronic musculoskeletal disease, affecting up to 30% of people with psoriasis. Research into PsA pathogenesis has led to the development of targeted therapies, including Tumor Necrosis Factor inhibitors (TNF-i). Good response is only achieved by ~60% of patients leading to ‘trial and error’ drug management approaches, adverse reactions and increasing healthcare costs. Robust and well-validated biomarker identification, and subsequent development of sensitive and specific assays, would facilitate the implementation of a stratified approach into clinical care. This review will summarise potential genetic biomarkers for TNF-i  response that have been reported to date. It will also comment upon the importance of managing clinical confounders when understanding drug response prediction. Variants in multiple gene regions including  A chronic inflammatory joint disease, psoriatic arthritis (PsA) has a prevalence of up to 30% in patients with psoriasis (Ps) , 2. It iAs the knowledge of psoriatic immunopathogenesis improved over the past 20 years, the therapeutic advancement of biologic drugs has also expanded dramatically. Drugs targeting Tumour Necrosis Factor (TNF) and IL-12/23/17 pathways, key in disease pathophysiology, have been developed . TNF is There are currently five TNF-inhibitor (TNF-i) drugs available for the management of PsA that have been approved by the Food and Drug Administration. Two classes of drug exist: TNF targeting monoclonal antibodies , infliximab (IFX), golimumab, and certolizumab pegol) and TNF fusion protein receptor inhibitors (etanercept (ETN)). Additionally, ADA, IFX and golimumab are full-length bivalent monoclonal antibodies while certolizumab pegol is a humanized IgG1 monoclonal antibody with a Fab’ fragment conjugated to polyethylene glycol. ETN is a genetically modified soluble fusion protein made of two extracellular portions of p75 TNFR linked to human IgG1 Fc . Both clh2 = 0.41)  (ref. ). HeritaPredicting which patients respond to a drug will save time, money, and debilitation in a complex condition, such as PsA, where there are multiple influencers of treatment response. To assess the utility of genetic predictors of response, all other clinical predictors also need to be acknowledged and understood.n = 1750) with Charlson comorbidity index >2 had higher baseline disease activity, increased occurrence of depression and anxiety and shorter TNF-i persistence compared to Charlson comorbidity index = 0 [n = 19,372), almost a quarter of all Ps and PsA patients  were obese, and TNF-i were less likely to be effective in obese patients [Female sex has been negatively associated with drug persistence and EULAR response in PsA cohorts across multiple TNF-i drugs and is considered the most consistent predictor of response , 29–33. ndex = 0 . Anxietyndex = 0 . Howeverndex = 0 . Lifestyndex = 0 , 36, 42.ndex = 0 , 43–45. patients . Overallpatients –48. Durapatients , 49, 50.patients .n = 765) reported a decline in efficacy from first to third biologic (68% vs. 37% moderate EULAR response), although conflicting results still remain [Choice of therapy may influence drug response, although results remain inconclusive. Some studies report no differences in persistence rates between first-line biologics ; one foul remain , 37, 41.l remain . PsA patl remain , 42, 51,l remain , 49, 52.n = 10), the development of ADAb was reported in 50% of IFX patients, 31% of ADA and none in ETN patients. Patients with anti-drug antibodies were more likely to be classified as ‘non-responders’, have lower drug levels and higher rates of drug inefficacy and withdrawal. Combination therapy with MTX showed a protective effect, while female sex was associated with higher levels of antibodies to TNF-i monotherapy; this supports the idea of females experiencing higher levels of immunogenicity [The development of anti-drug antibodies (immunogenicity) has been associated with reduced efficacy to monoclonal TNF-i therapy in PsA –55. In agenicity . TNF-i tgenicity , 58. Furgenicity .Overall, understanding the contribution of clinical factors and their association with drug response is imperative. This will allow the appropriate and personalised management of some of these factors, including adherence, comorbidities, and mental health conditions. Furthermore, it is realistic that the development of any prediction models of pharmacogenetic drug response will include well validated and identified clinical markers of response.Due to the highly varied interindividual biologic efficacy and response to treatment, there is great potential to improve the management of PsA patients therapy via the identification of predictive biomarkers . DNA varTNF-a) for association with response to ADA, given that the gene is the target of the drug. Part of the major histocompatibility complex class III region on the shorter arm of chromosome 6, the gene is located between HLA-B and HLA-DR [n = 57) evaluating the role of TNF-a polymorphisms with different TNF-i response, a variant in the first intron of the gene was associated with both first- or second-line ADA responders [TNF-a + 489 A/A genotype (rs80267959) was observed in responders (n = 5/13) but not in non-responders (n = 0/3) , +489 (rs80267959) and −238 (rs361525), with the first polymorphism to be associated with PsA drug response identified at position −308 (Table n = 86) the −308 G/G genotype was associated with increased response to ETN independently, as well as in combination with other TNF-i (ADA and IFX) [n = 57), the  + 489 TNF-a promotor was reported to not only be associated with PsA disease severity and development, but also with ETN response [TNF-a + 489 G/G and G/A genotypes were observed at an increased frequency in responders compared to non-responders, while all A/A patients demonstrated no response, although this finding failed to reach statistical significance. In contrast, association was noted with the −238 variant (rs361525) and ETN response. Further lack of evidence for the influence of −238 and −308 polymorphisms on TNF-i response has also been reported [n = 10–103) which impacts power to detect an association.ETN is a dimeric fusion protein that acts as a TNF-α receptor inactivating TNF following binding. Due to its role in PsA aetiopathogenesis g −308 rs800629, +and IFX) . Under aand IFX) . Of noteand IFX) . However −238 rs3525, withand IFX) , 67. In response . TNF-a +reported , 68. Thereported , 69. Altreported . StudiesFCGR2A-131 and FCGR3A, on response has been investigated. In PsA patients (n = 103) prescribed ETN (~53%), IFX or ADA, higher affinity alleles, in either the homozygous or heterozygous state, variants in FCGR2A-131 (rs1801274) associated with a higher 6 month response [FCGR3A polymorphisms showed an association in any drug, which is supported by other PsA studies [FCGR2A-131 high affinity findings contradict those in RA where low affinity alleles are linked to a better response to IFX therapy [The fragment crystallizable region of TNF-i drugs, including ETN, binds to Fc-y receptors and so the effect of polymorphisms in genes influencing the affinity to this region, response . Followi studies . Interes therapy .n = 20) reported that rs610604 and rs6920220 in TNFAIP3 are significantly associated with quality of life improvement at both the 3 and 6-month stages [Furthermore, when investigating the effect of TNF-i response collectively, a modest Spanish PsA cohort (h stages . This isTNF-R1A/TRAIL1/FCGR3A), with various levels of validation in different cohorts (Table TRAILR1) and Tumour Necrosis Factor Receptor 1 A (TNF-R1A/TNF-R1/TNFRSF1A) have been associated with both chronic inflammatory diseases and cancer susceptibility, thus these death receptors are thought to play an important role in immune system homeostasis. TNF-R1A rs767455 was associated with a significantly improved 3-month EULAR response in 55 PsA patients with the homozygous A/A genotype versus the homozygous G/G or heterozygous A/G genotype, with the A allele significantly associated with a better response. Of interest, this demonstrated an opposing effect to that seen at 3 months in RA patients, potentially highlighting a distinct difference in pathophysiology between the diseases [TNFRSF1A) rs1800693, rare G/G genotype, was associated with delayed response to IFX but not ADA therapy. Response was assessed using The Bath Ankylosing Spondylitis Disease Activity Index [TRAILR1/TNFRSF10A (8p21) is part of the TNF-receptor superfamily and is involved in cell apoptosis induction. The association of TRAILR1 polymorphisms was investigated in a mixed cohort of RA and PsA. In a subgroup analysis of 27 IFX-treated PsA patients, patients with rs20575 C/C genotype in TRAILR1 demonstrated an improved 6 month EULAR response to IFX [FCGR3A encodes for the fragment crystallizable portion of immunoglobulin G and is a mediator of antibody cell mediated cytotoxicity. In one study rs396991 V158F higher affinity V/F and V/V genotypes were associated with a significantly improved 3 month EULAR response in PsA (n = 16) patients, perhaps due to increased immunoglobin binding and natural killer activation, although the small cohort size is of note [TRAIL-R1/ TNFRSF10A, FCGR3A).A chimeric monoclonal antibody, IFX binds and neutralizes TNF-a. Polymorphisms associated with response to IFX have been reported in multiple genes  ([The future of stratified medicine requires the identification of robust and validated biomarker panels. Genetic variants are attractive as potential biomarkers as they only need to be tested once; furthermore, in other areas of medicine, genetic variants have been reported to associate with treatment effects , 77, 78.PsA is a clinically heterogeneous disease, affecting the skin, axial and peripheral skeleton. Whilst several outcome measures exist, the majority have been developed initially in RA, a disease that is genetically distinct from PsA. In addition, most outcome measures used in PsA studies include a subjective assessment of response which may be affected by other diseases such as depression or chronic non-inflammatory pain. The lack of specific and objective PsA outcome measures for pharmacogenetic studies is a significant barrier to the identification of genetic biomarkers of response and should be an area for future research. Furthermore, future work should also focus on the association between genetic variants and drug response in specific disease domains, rather than non-representative disease activity composite scores. By investigating response in enthesitis, psoriasis or joint erosion domains, independently, the role of pharmacogenomics in PsA response could be elucidated, whilst reducing heterogeneity in PsA and enhancing opportunities for personalised medicine approaches.Whilst several clinical factors are associated with TNF-i response, none are of significant effect size to be used alone and it is likely that they will need to be combined with other biomarkers of response to make clinical decisions. To date, few pharmacogenetic studies have included clinical factors, such as adherence, in the identification of genetic biomarkers of response, perhaps due to the small sample size available.For these reasons, there are currently no validated clinically used genetic biomarkers of response in PsA. To successfully include genetic biomarkers in the development of predictive models of TNF-i response, there remains a need for hypothesis-free genome-wide association studies in large well-characterised cohorts where response to treatment has been consistently measured. Due to the prevalence of PsA, it is likely that such cohorts would need to be developed through international collaboration to improve sample size and power. Furthermore, the role of potential confounders of response, including ADAb, adherence, concomitant therapy and lifestyle factors should be considered. The identification and validation of genetic biomarkers of TNF-i response in PsA, would enable a stratified medicine approach to be adopted clinically, reducing economic burden and improving quality of life.


Thiothrix was observed, was sequenced. Representatives of the phyla Gammaproteobacteria, Cyanobacteria and Campilobacteriota dominated in the microbial community. The complete genome of Thiothrix sp. KT was assembled from the metagenome. It displayed 93.93–99.72% 16S rRNA gene sequence identity to other Thiothrix species. The average nucleotide identity (ANI) и digital DNA-DNA hybridization (dDDH) showed that the genome designated KT represents a new species within the genus Thiothrix, ‘Candidatus Thiothrix sulfatifontis’ sp. nov. KT. The taxonomic status has been determined of the strain Thiothrix sp. CT3, isolated about 30 years ago and not assigned to any of Thiothrix species due to high 16S rRNA gene sequence identity with related species . The complete genome sequence of strain CT3 was determined. The ANI between CT3 and other Thiothrix species was below 82%, and the dDDH values were less than 40%, indicating that strain CT3 belongs to a novel species, Thiothrix winogradskyi sp. nov. A genome analysis showed that both strains are chemo-organoheterotrophs, chemolithotrophs (in the presence of hydrogen sulfide and thiosulfate) and chemoautotrophs. For the first time, representatives of Thiothrix showed anaerobic growth in the presence of thiosulfate.The metagenome of foulings from sulfidic spring “Serovodorodny” , where members of the genus  Thiothrix, first described by Winogradsky in 1888. Representatives of this genus have similar morphotypes, lead an attached lifestyle, form rosettes, mucous sheaths (only Thiothrixunzii A1T lacks a mucous sheath) and are able to accumulate elemental sulfur intracellularly during lithotrophic growth in the presence of reduced sulfur compounds . Th. ThThiotd of 95% . The obtThiothrix sp. KT with other members of the genus Thiothrix were in the range 93.93–99.72% , who described the genus Thiothrix). ® 80, phenol, butyrate, n-amyl alcohol, oleic acid, benzoate, butanol, D-(+)-glucose, lactose, maltose, citrate, ethanol, methanol, propanol and glycerol. The CT3 strain is capable of using ammonium, nitrite, nitrate and urea as nitrogen sources. Oxidase-positive, catalase-negative. Capable of dissimilatory nitrate reduction: nitrate is reduced to N2O. It stores polyhydroxybutyrates and sulfur intracellularly. Oxidizes thiosulfate to sulfate and elemental sulfur  cluster; and phsC, which encodes a membrane anchor protein, participating in the transfer of electrons from the quinone pool. The catalytic PhsA subunit contains the N-terminal Tat signal peptide indicating its location on the outer side of the membrane. Such complexes could be responsible for the reduction of various electron acceptors, including thiosulfate, tetrathionate, nitrate and arsenate [Ca. Thiothrix sulfatifontis’ KT genes encoding potential thiosulphate reductases were found in the genomes of other members of the genus Thiothrix: T. fructosivorans QT, T. caldifontis G1T, T. lacustris BLT, T. litoralis AST, T. nivea JP2T, T. unzii A1T, MAG of Thiothrix sp. 207, “Ca. Thiothrix anitrata” A52, T. subterranea Ku-5T. For some members of the genus, anaerobic growth on thiosulfate (about 15 mg protein/l) was shown with the formation of sulfide and sulfite, products characteristic of the reduction of thiosulfate with the participation of thiosulfate reductase: T. caldifontis G1T, T. lacustris BLT, T. litoralis AST, T. nivea JP2T, T. unzii A1T.Dissimilatory sulfur metabolism in  sulfate . The oxiarsenate . BesidesThiothrix among proteobacteria [Thiothrix. This was probably an unusual factor for the formation of a new species of the genus. It was possible to assemble the MAG Thiothrix sp. KT from the metagenome of microbial fouling of the “Serovodorodny” spring, which was assigned to a new species, ‘Ca. Thiothrix sulfatifontis’ sp. nov. KT, according to its ANI and dDDH values.Unique, unusual habitats are often a source of new, previously undescribed isolates. An analysis of the metagenome of the microbial community of sulfidic spring “Serovodorodny”  showed that its composition is typical for microbial mats of a sulfide spring developing under natural light, i.e., the predominance of proteobacteria and cyanobacteria in the above-ground sulfur microbial mats and the dominance of bacteria of the genus bacteria . HoweverThiothrix sp. CT3 was obtained. Its analysis made it possible to describe the strain as a representative of a new species—Thiothrix winogradskyi sp. nov. CT3.In addition, during this work a whole genome sequence of the strain Ca. Thiothrix sulfatifontis’ KT does not encode genes for dissimilation nitrate reduction, which was previously shown only for ‘Ca. Thiothrix anitrata’ [Thiothrix, currently available in GeneBank, made it possible, for the first time, to identify the phsABC genes of dissimilatory thiosulfate reductase in T. fructosivorans QT, T. caldifontis G1T, T. lacustris BLT, T. litoralis AST, T. nivea JP2T, T. unzii A1T, MAG of Thiothrix sp. 207, ’Ca. Thiothrix anitrata’ A52, T. subterranea Ku-5T, ‘Ca. Thiothrix sulfatifontis’ KT, except for T. winogradskyi CT3T and ‘Ca. Thiothrix moscowensis’ RT. Representatives of the genus Thiothrix, isolated in pure culture and in which the phsABC genes encoding thiosulfate reductase were found, were capable of anaerobic growth in the presence of thiosulfate.In both genomes, genes encoding enzymes of the main metabolic pathways of energy and constructive metabolism, providing organo-heterotrophic, chemolithoheterotrophic and chemolithoautotrophic growth, were found. However, metabolic features have been identified. In particular, the ‘nitrata’ . Also, aThiothrix: ‘Candidatus Thiothrix sulfatifontis’ sp. nov. KT and Thiothrix winogradskyi sp. nov. CT3T. This study describes two new species within the genus Thiothrix sp. KT was assembled from the metagenome of the microbial community of the “Serovodorodny” sulfidic spring .MAG Thiothrix sp. CT3 was not determined as a species due to the low information content of its 16S rRNA gene as a phylogenetic marker. In this work, the complete genome sequence of strain CT3 was determined. Phylogenomic analysis and the use of similarity indexes for genomic comparisons, i.e., AAI, dDDH and ANI, made it possible to assign strain CT3 to a new species, namely, Thiothrix winogradskyi.For more than 30 years,