Wounds on embryonic mouse fetuses regenerate up to embryonic day (E) 13, but after E14, the pattern is lost and a visible scar remains. We hypothesized that the sonic hedgehog (Shh), which is involved in patterning during development, is involved in the regeneration of texture. Embryos of ICR mice were surgically injured at E13, E14, and E15 and analyzed for the expression of Shh. For external Shh administration, recombinant Shh-containing slow-release beads were implanted in the wounds of mice. In contrast, cyclopamine was administered to wounds of adult mice to inhibit Shh. The expression of Shh was unaltered at E13, whereas it was upregulated in the epidermis of the wound from E14 onward. Implantation of recombinant Shh-containing beads into E13 wounds inhibited skin texture regeneration. Cyclopamine treatment inhibited epithelialization and thickening of the epidermis in the wounds of adult mice. In vitro, Shh promoted proliferation and inhibited the migration of epidermal keratinocytes through the activation of cyclin D proteins. Thus, our results suggested that the expression of Shh is involved in the regeneration of texture during wound healing, especially in epidermal keratinocyte migration and division, and could inhibit skin texture regeneration after E14. Monodelphis domestica [Fetal skin wounds made at certain developmental stages regenerate completely without scarring. For example, wounds inflicted on mouse fetuses on gestational (E) days 13–16 are completely histologically healed [omestica . One of Complete skin regeneration requires the restoration of the surface structure, as of well as the dermis and skin appendages. Therefore, it is necessary to delineate the difference in the wound-healing process after E13 and E14, which is the switch between the presence and absence of a visible scar on the skin. The interaction between the epidermis and the dermis was reported to be maintained at E13, which could regenerate texture [One factor involved in patterning is hedgehog protein. Hedgehog, an important determinant of cell fate, is involved in the patterning of the skeleton and organs during the development of various organisms ,5. SonicA recent study on Shh and wounds reported that the regeneration of new hair follicles during the healing of major skin excisions in mice involves Shh signaling that plays a major role in the conversion of wound fibroblasts from scar-promoting to hair follicle neogenesis stimulated fibroblasts. This phenomenon can be referred to as true tissue regeneration ,10,11.This link between Shh and hair follicle regeneration, a true tissue regeneration, has attracted widespread interest, but its relevance to complete skin regeneration, including texture, remains unclear.We hypothesized that Shh can determine epidermal patterning and could be involved in the transition from complete regeneration of epidermal patterns in E13 wound healing to visible scar formation in late fetal wound healing.We analyze Shh expression of Shh in the epidermis during fetal wound healing and investigated the effect of Shh on the formation of skin furrows and skin papules. We focused on the 12- and 24 h post-injury timepoints of the wound-healing process, as fetal mouse wounds heal quickly, completing epithelialization in approximately 48–72 h [The research protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Keio University School of Medicine . All experiments were conducted according to the institutional guidelines for animal experiments at the Keio University. This study was reported in accordance with the Reporting of In Vivo Experiments on Animals (ARRIVE) guidelines.Eight-week-old ICR mice were obtained from Sankyo Laboratories . Embryos with a vaginal plug observed in the maternal vagina were designated E0. Surgery was performed on E13, E14, and E15 fetuses. Pregnant maternal-fetal mice were anesthetized with 4% isoflurane , which was maintained at 2% during surgery. The surgical site was sterilized with 70% ethanol, the myometrium and amnion were opened, and a 2-mm-long full-layer incision was made in both flanks of the fetus using sterile microsurgical scissors  (n = 12 per timepoint). In E13 and E14, after wound creation, only the amniotic and yolk sacs were sutured with 9-0 nylon threads, whereas the myometrium was left open and unstitched. In E1, after wounding the fetus, only the myometrium was sutured with 9-0 nylon threads, the uterus was returned to the abdominal cavity and the abdomen was closed. Before closing the abdomen, 1 μg ritodrine hydrochloride  until sectioning.Ten-week-old male ICR mice were anesthetized with isoflurane and two full-layer wounds (approximately 1 cm in diameter) were created per mouse on both sides of the chest using a No. 11 scalpel on the shaved back skin. This procedure was performed on five adult mice. At 0, 12, 24, 72, and 120 h after wounding, the mice were euthanized by cervical dislocation. The wounds were then excised, harvested and fixed overnight in 4% paraformaldehyde, soaked in 20% sucrose/phosphate-buffered saline (PBS), embedded in OCT compound  and cut into 7-μm–thick sections.Mouse recombinant Shh  was dissolved in PBS to make a 1 µg/µL Shh solution. Cross-linked agarose beads  were immersed in PBS for 24 h to prepare Shh-soaked beads. The control beads were soaked in bovine serum albumin (1 µg/µL) dissolved in PBS and placed in the wound of E13 fetuses. The fetuses were collected after 72 h, Indian ink was dripped onto the skin, and the texture was observed under a stereomicroscope.Ten-week-old male ICR mice were anesthetized with isoflurane inhalation. The wounds were then treated with 100 µL drops of cyclopamine  at various concentrations  dissolved in PBS and protected with a clear film once daily. The control wounds were treated with 100 µL of PBS dropwise. After 7 d, the animals were euthanized by cervical dislocation and the wounds were excised and collected. The tissues were fixed by immersion in 4% paraformaldehyde overnight. The samples were embedded in paraffin and cut into 7-µm-thick sections.® M.O.M. kit . After washing three times with PBS, slides were incubated with mouse monoclonal Shh antibody . Subsequently, after washing thrice with PBS, slides were incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody  for 1 h at room temperature, washed three times with PBS, and then embedded in ProLong gold (Thermo Fisher Scientific). The slides were then observed under an all-in-one stereomicroscope . The relative intensity of the fluorescent signal was quantified as arbitrary units (AU) by measuring the average fluorescent intensity of a 10-µm2 sized area at a minimum of 10 locations in the wound. Data from uninjured tissue were subtracted to remove nonspecific background fluorescence.Paraffin-embedded specimens were cut into 7-μm-thick sections and mounted on glass slides. After drying overnight at room temperature to allow specimens to adhere to slides, paraffin was dissolved in a slide heater  at 65 °C for 30 min immediately before use. The slides were then deparaffinized by immersion in xylene twice at room temperature (5 min each). Slides were transferred twice to 100% ethanol (3 min each), once to 95%, 70%, and 50% ethanol (3 min each) and rehydrated at room temperature. Subsequently, 1.5% bovine serum albumin was used for blocking for 1 h at room temperature, and mouse primary antibodies were localized on mouse tissue using the Vector4 cells were seeded on a 96-well plate and treated with 2% fetal bovine serum , 50 μg/mL recombinant Shh + 2% FBS, or 50 μg/mL cyclopamine + 2% FBS. Cells were incubated at 37 °C for 48 h, and cell proliferation was evaluated using the CellQuanti-MTT Cell Viability Assay Kit .The PAM212 mouse keratinocyte cell line was obtained from Thermo Fisher Scientific. Briefly, 1 × 104 PAM212 was seeded on a plastic dish and grown to confluence. Cells were scratched with a cell scraper with a width of 500 µm. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 50 μg/mL of recombinant Shh or cyclopamine  for 24 h. The edges of each scratch were examined under a microscope and the wound area was measured using ImageJ software . The experiment was independently repeated three times.Briefly, 1 × 10The wounded skin was collected under a stereomicroscope by cutting as close to the wound margin as possible. A monophasic solution of phenol and guanidine isothiocyanate  was used to extract total skin RNA from both injured and uninjured specimens according to the manufacturer’s instructions. The cDNA synthesis by reverse transcription was performed after mixing the extracted total RNA with random primers, reverse transcriptase, and dNTP mixture .-ΔΔCt method.RT-qPCR and transcript quantification were performed on an Applied Biosystems 7500 Fast real-time PCR system (Thermo Fisher Scientific) using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) in different samples (in triplicate) with the following primers for the quantification of transcript levels: Shh (Mm00436528_m1), patched1 (Mm00436026_m1), Gli1 (Mm00494654_m1), Gli2 (Mm01293116_m1), Gli3 (Mm00492337_m1), Cyclin D1 (Mm00432359_m1), Cyclin D2 (Mm00438070_m1). The housekeeping gene ACTB (Mm02619580_g1) was an endogenous normalization control. The level of gene expression in the proliferating cell population was a baseline and the fold change values were determined using the 2In situ hybridization analysis was performed using the QuantiGene ViewRNA ISH Tissue Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, paraffin sections were dried at 60 °C for 60 min and paraffin removal was performed with Histo Clear  and 100% ethanol with an ImmEdge Pen. After washing twice with PBS, the tissue was fixed in 10% neutral buffered formaldehyde solution for 5 min and washed again with PBS. The target probe was diluted 50-fold in probe set diluent QF solution heated to 40 °C and incubated at 40 °C for 3 h. After washing three times with wash buffer, the probe was incubated in a preamplifier solution at 40 °C for 25 min. Following another wash with wash buffer three times, the probe was incubated in a preamplifier solution at 40 °C for 15 min. The AP enhancer solution was decanted and the Fast Red Tablet was dissolved in Napthol buffer and incubated at room temperature for 5 min. After decanting the AP enhancer solution, the Fast Red Tablet was dissolved in Napthol buffer and incubated at 40 °C for 30 min. After being washed twice with PBS, nuclear staining was performed with Gill’s Hematoxylin solution and washed three times with water. The probe used was Shh (VB6-13424-VC).U tests were performed to determine differences in migration or gene expression using Statistica software version 9.0 . All values are presented as mean ± SD. The threshold for statistical significance was established at p < 0.05. Each experiment was conducted in triplicate.Mann–Whitney First, we investigated the expression of Shh in mouse wounds during development from embryonic to postnatal stages. We found that Shh was expressed in the epidermis of adult mice at all timepoints, 1, 3, 5, and 7 d after wounding A, particUsing RT-PCR, we detected that the expression of Shh and all signaling molecules involved in canonical Shh signaling, such as patched1, Gli1, Gli2, and Gli3, was downregulated in the wounds of E13 mice compared to normal skin. However, the expression of Shh and signaling molecules downstream of patched1 increased in the wound as the developmental stage progressed D.Next, we investigated the effect of suppressing Shh signaling on wound healing in adult mice. We administered cyclopamine, an inhibitor of the Hedgehog signaling pathway, to wounds of adult mice daily. To determine how cyclopamine affects wound healing in adult mice, two concentrations  were administered. Therefore, epithelialization was suppressed in mice treated with high concentrations A. FurtheTo investigate the possibility that Shh signaling might be involved in regeneration of skin texture regeneration, we implanted slow-release beads soaked in recombinant Shh in the wounds of E13 mice and expected that they should completely regenerate skin texture A. We fouTo investigate the mechanism of the epidermal thickening observed in vivo, we examined the proliferative and migratory capacity of epidermal cells in the presence of Shh inhibitors and recombinant Shh. We found that recombinant Shh promoted the proliferation of epidermal cells, whereas cyclopamine treatment inhibited their proliferation A. FurtheEpidermal–dermal interactions and patterning factors might be involved in the processes leading to loss of complex skin texture during wound healing; however, the underlying mechanisms of these regenerative processes have not been elucidated. We observed the expression of Shh, a factor involved in patterning and hair follicle formation, during the development of fetal mouse and in wound healing in adult animals. We also examined the effects of its expression on skin texture formation.Although Shh signaling is closely involved in hair follicle development and skin tumorigenesis, the underlying mechanisms and downstream events it coordinates on the skin remain elusive. Here, we observed that Shh signaling was downregulated in the wounds of E13 mice with regenerating skin texture, and external Shh supplementation inhibited texture regeneration.In our previous study, we reported that complete skin regeneration, including skin texture, requires epidermal and dermal positioning . At E13,Shh plays an important role in both fetal and adult hair development, and in mice lacking Shh, mature hair follicles do not develop, despite the initiation of follicle formation and the formation of dermal condensates ,14,15. FShh and Gli2 mutant skin and are induced by the activator function of Gli2 [In various developmental situations, Hedgehog signaling is associated with proliferative responses of target cells. For example, quiescent cells are stimulated to enter the cell cycle in response to mitogenic signals such as Shh that induce the expression of cyclin D proteins, which is required to pass the G1 restriction point . Shh sig of Gli2 , no embrWe also provided evidence that the Shh pathway might regulate keratinocyte motility. In a previous report, overexpression of Shh induced the invasion of HaCaT cells, which depended on an intact EGF signaling axis, as opposed to the inhibition of neural crest cell motility . AlthougThe Hedgehog pathway includes canonical signaling mediated by Smoothened (Smo) activation and induces stabilization and activation of the Gli family of potential zinc finger transcription factors; in contrast, there is non-canonical signaling only via patched, which functions as a dependent receptor independent of Smo through regulation of cyclin B1 and caspase-9 . The ShhA limitation of our study was that we found a contradiction in that downregulation of Shh signaling is necessary for skin texture regeneration but detrimental to hair follicle regeneration. Thus, simply downregulating Shh to achieve a wound-healing scheme that regenerates the skin, including its texture, as in E13, leads to inhibition of hair follicle formation. Therefore, it will be necessary to create mice with epidermal cell-specific conditional knockout of Shh or knockout of downstream targets of Shh signaling—cyclin D and Gli family members, and observe the phenotypic changes in skin texture.In this study, we showed that Shh signaling was altered during the healing of fetal mouse wounds at E13 and E14 and beyond, specified the transition point for texture regeneration, and revealed that Shh affected the motility and proliferative capacity of epidermal keratinocytes. These findings could contribute to the development of methods for the regeneration of skin texture and the healing of scars without a visible mark.


Pancreatic cancer remains the third leading cause of death amongst men and women in the United States. Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer maintains its reputation of being the most aggressive with a poor prognosis. One of the contributing factors to the high mortality of PDAC is the absence of biomarkers for early detection of disease and the complexity of tumor biology and genomics. In this review, we explored the current understanding of epigenetics and diagnostic biomarkers in PDAC and summarized recent advances in molecular biology. We discussed current guidelines on diagnosis, prognosis, and treatment, especially in high-risk individuals. We also reviewed studies that have touched on identifying biomarkers and the role they play in making early diagnosis although there are currently no screening tools for PDAC. We explored the recent understanding of epigenetic alterations of PDAC and the future implications for early detection and prognosis. In conclusion, the new and emerging advances in the detection and treatment of PDAC can lead to an improvement in the current outcome of PDAC. The incidence of PDAC increases steadily by decade after age 40, classically being diagnosed in the 7th decade, with only about 10% of cases in individuals under 55 years [. Pancreatic cancer is the third leading cause of death in men and women. The worldwide five-year survival for pancreatic cancer ranges from 2-9% [Despite years of research and advances in understanding the disease mechanisms, Pancreatic Ductal Adenocarcinoma (PDAC) maintains its reputation as an aggressive disease with a poor prognosis, accounting for almost as many deaths as new cases yearly, according to the Global Cancer Incidence, Mortality and Prevalence (GLOBOCAN) statistics . The inc55 years ,3. Pancrrom 2-9% .. Other factors contributing to the high mortality of PDAC include the absence of biomarkers for early disease detection and the complexity of tumor biology and genomics, limiting the effectiveness of conventional medical therapy [ There are also complexities in the genetic mutations associated with PDAC; for instance, the pattern of the major genetic mutation seen in PDAC, that is, the activation of the oncogene KRAS is distinct from other malignancies in that KRAS mutation is an inciting event in PDAC, and is seen in the precursor lesions, Pancreatic intraepithelial neoplasia (PanIN). In contrast, it emerges as a late oncogenic driver after epigenetic changes have been created by founder mutations in other malignancies [TP53 and CDKN2A, and attempts at targeting therapy against these mutations have proved futile [The United States has a five-year relative survival rate of 11%, the lowest amongst solid tumor malignancies . This co therapy ,10. Thergnancies . Other gd futile . Considering these, it goes without saying that to improve the prognosis of PDAC, we need early diagnostic biomarkers and other modalities of treatment that target epigenetic alterations. Therefore, this review explores the current understanding of epigenetics and diagnostic biomarkers in PDAC. It also summarizes recent advances in molecular biology and their therapeutic and prognostic implications in pancreatic cancer.MethodologyWe employed a preset standard for our data search, selection, and inclusion. The search was from the repositories of PubMed, Google Scholar, and ScienceDirect Library. Articles included were from the last ten years, i.e., 2012-2022. They included randomized clinical trials, meta-analyses, systematic reviews, and other observational studies. Our keywords for the search were: (tumor markers) (biomarkers multiomics) (pancreatic cancer). We used the search engine layout to generate all possible pieces of literature with these word combinations. The google scholar search initially returned a total of 6,830 results; To trim down to the most specific articles, we employed the ‘advance search’ on google scholar. The advanced search using ‘tumor marker’ for ‘exact word’ resulted in 51 articles, ‘biomarkers in pancreatic cancer’ returned 44 article search results, and ‘multi-omics in pancreatic’ returned three article results. Using the same ‘advanced search’ approach, we searched PubMed and ScienceDirect and found 32 and 17 related articles, respectively.Eligibility CriteriaA total of 147 articles were screened by five independent contributors, first using the contents of the abstract and then a full-text read. A conflict of inclusion or exclusion between the contributors was settled by the vote of the fifth contributor. We further narrowed the articles to 31, and we analyzed the studies according to the various facets of the interest of our study. A concise inclusion and exclusion criteria are shown in Table Genetics and epigenetics in pancreatic cancerKRAS, TP53, SMAD4, and CDKN2A) and also identified two additional genes not previously described in human pancreatic ductal adenocarcinomas (KDM6A and PREX) [MMPS) 7 and integrin, alpha 2 which are significantly different in normal tissues versus tumor samples and can be used in diagnosis and therapy in the future. They stated that MMPS is involved in cell proliferation, migration, and differentiation and may correlate with cancer activity and poor prognosis. The interaction between them and extracellular ligands induced a signaling cascade that regulated intracellular and extracellular activities [Various studies have been carried out to identify genes that play a role in pancreatic cancer. A study was carried out by Waddell et al., in which the genome sequencing of 100 pancreatic ductal adenocarcinomas was done . They cond PREX) -13. Anotnd PREX) . They intivities -16.Epigenetics refers to the changes in the phenotype which affect gene expression without altering the DNA sequence. A study by Khoshchehreh et al. emphasizes the role of epigenetics reprogramming in the tumorigenicity of pancreatic adenocarcinoma . Their rEpigenetic changes are reversible, making them targets for tumor-directed therapies. They occur through the methylation of DNA, histone modifications, and changes in chromatin structures. Epigenetic regulation and the corresponding changed chromatin states frequently occur in genes partaking in oncogenic signaling, metabolic alterations, and the metastatic process. ,20-26. TUSP22) and stromal desmoplasia driven by focal adhesion kinase (FAK). Increased lactate levels and suppression of lysine demethylase 3A (KDM3A) are thought to be the mechanism for decreased T-cell infiltration [Epigenetic modifications are also responsible for the heterogeneous tumor microenvironment in PDAC. The changes drive tumor desmoplasia and induce the creation of a hypoxic and immunosuppressive tumor microenvironment (TME) with a lack of T-cell infiltration and resistance to immune-checkpoint inhibition (ICI) therapy. These immune modifications are thought to be a result of transcriptional regulations driven by the activation of ubiquitin-specific protease 22 , and MRI ,23. SurgIn its updated guidelines, the International Cancer of the Pancreas Screening Consortium came to a consensus that screening for pancreatic cancer in patients with a familial risk should begin no earlier than 50 or 10 years earlier than the youngest relative with pancreatic cancer. MRI/magnetic retrograde cholangiopancreatography (MRCP) and/or endoscopic ultrasound (EUS) were the preferred diagnostic modalities ,28. The Future trends in diagnosis and prognostication of pancreatic adenocarcinomaADAMTS1 and BNC1 to determine the usefulness of this two-gene panel as a non-invasive biomarker set for the early detection of pancreatic cancer. The two-gene panel (ADAMTS1 and/or BNC1) was positive in 100% (8/8) of stage I, 88.9% (8/9) of stage IIA, and 100% (20/20) of stage IIB disease. The sensitivity and specificity of the two-gene panel for localized pancreatic cancer (stages I and II), where the cancer is eligible for surgical resection with curative potential, was 94.8% and 91.6%, respectively [A more recent understanding of epigenetic alterations and the heterogenous tumor microenvironment of pancreatic adenocarcinoma have paved the way for promising biomarkers with potential for diagnosis and predicting both survival and response to therapeutic interventions. These include selectively expressed DNA methylation biomarkers, circulating and tissue-bound transcriptomes, and fecal microbiota signatures. Eissa et al. used circulating cell-free DNA (cfDNA) from a large cohort of patients at different stages of pancreatic cancer and an age-matched normal group to study DNA methylation of genes ectively ,21,28. TCirculating transcriptome contains diverse non-coding, stable and functional elements such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Their resistance to RNase activity and readiness to be detected in the biological fluids of cancer patients showcase their potential as biomarkers in PDAC. Profiling miRNA expression can correlate to the stage of malignant pancreatic disease and hold potential as diagnostic and prognostic markers ,23,27. VAn increased ratio of mIR-3940-5p/miR-8069 detected in urine exosomes has been reported in the early stages of PDAC. With improved diagnostic accuracy when combined with CA19-9  ,27. SomeAlso, high expression levels of HOTAIR and HK2 have been associated with significantly lower overall survival rates than patients with low expression levels. Conversely, low expression levels of lncRNA LINC01111, which acts as a tumor suppressor, are associated with a poor prognosis ,23,27. MMET, B3GNT3, SPAG4, RPE, KIF20A, CDK1, PGK1, AURKA, P4HA1, PYGB, HMMR, SDC1, and EFNA3. Based on these results, they developed a three-gene signature  and risk score for prognosis prediction of PDAC. Patients were divided into high-risk and low-risk groups based on their scores. The overall survival (OS) was significantly poorer in the high-risk group than in the low-risk group (p < 0.000) The area under the curve (AUC) for one-year OS was 0.764. They also found that when combined with other variables like age, gender, tumor stage, radiotherapy, and tumor residual, the scores obtained predicted survival time (C-index 0.67 {SE}=0.031) in a way that showed potential suitability for clinical application [Research into the tumor microenvironment and metabolic pathways associated with the progression of pancreatic cancer has opened up a new direction for diagnosis and prognosis in patients with pancreatic adenocarcinoma . Li et alication ,27.Karasinka et al., in another study, investigated metabolic variants of PDAC using RNA data from resectable and nonresectable tumors to determine the relative expression of the cholesterol and glycolytic pathway genes in a quest to determine their relationship with OS. They stratified them into four metabolic subgroups: quiescent, glycolytic, cholesterogenic, and mixed. They also noted significant differences in survival and metastatic potential between the subgroup. The glycolytic profile was associated with the shortest median survival in both resectable (log-rank test p=0.018) and metastatic (log-rank test p=0.027). Interestingly, a survival benefit was observed in cases with increased expression of cholesterol synthesis genes. Cholesterogenic cases had the longest median overall survival in the resectable  and metastatic (log-rank test p=0.011 vs. glycolytic) groups. Cholesterogenic cases had the longest relapse interval in resected PDAC . Thus, mThis review highlights recent advances in molecular biology and the therapeutic and prognostic implications in pancreatic cancer. With advances in technology over the last 10 years, there have been new biomarker discoveries geared toward improving the outcome of patients with PDAC. We discovered that the recent understanding of the epigenetics of PDAC had paved the way for promising biomarkers with diagnostic and prognostic value. Researchers have found that DNA methylation-based biomarkers, transcriptomes, metabolic gene profiles, and fecal microbiomes are valuable in the early detection, diagnosis, and prognosis of PDAC. We also found that some newly discovered biomarkers can detect tumor aggressiveness and predict overall survival. These new advances in early detection should inform scientific research geared towards improving overall outcomes in PDAC patients.


Understanding molecular epidemiology is essential for the improvement of lumpy skin disease (LSD) eradication and control strategies. The objective of this study was to perform a molecular characterization and phylogenetic analysis of lumpy skin disease virus (LSDV) isolated from dairy cows presenting LSD-like clinical signs in northern Thailand. The skin nodules were collected from 26 LSD-suspected cows involved in six outbreaks during the period from July to September of 2021. LSDVs were confirmed from clinical samples using the polymerase chain reaction (PCR). The PCR-positive samples were subsequently amplified and sequenced using a G-protein-coupled chemokine receptor (GPCR) gene for molecular characterization and phylogenetic analyses. All 26 samples were positive for LSDV by PCR. A phylogenetic analysis indicated that the 24 LSDV isolates obtained from cattle in northern Thailand were closely related to other LSDV sequences acquired from Asia . On the other hand, two LSDV isolates of the cows presenting LSD-like clinical signs after vaccination were clustered along with LSDV Neethling-derived vaccines. The outcomes of this research will be beneficial in developing effective control strategies for LSDV. Capripoxvirus in the family Poxviridae and is known to cause lumpy skin disease (LSD) [The lumpy skin disease virus (LSDV) belongs to the genus se (LSD) . LSD is se (LSD) . It has se (LSD) .LSD was discovered in Zambia in 1929 and then spread to most areas of Africa. Consequently, the disease gradually spread to the Mediterranean basin, Europe, Middle East, and Asia ,5. In 20Eradication and control measures for LSD are comprised of cattle movement restrictions, the dissemination of vaccinations, and disinfection and arthropod control protocols . A live-Understanding molecular epidemiology is necessary for the improvement of LSD eradication and control strategies . GeneticThailand is predominantly an agricultural country with a cattle population of approximately 10 million heads. Most of farms are small-holders with a herd size of 20–30 animals per farm. Animals are kept in open-yard housing with semi-intensive managements. The cattle are mostly raised for domestic consumption. However, it has been reported that about 100,000 heads of large ruminants were exported to neighboring countries . The fir®; Vetal Animal Health Product SA; Adiyaman; Turkey). Furthermore, 1 sample of a commercial vaccine which was used in those farms was also collected for the PCR and the GPCR gene sequencing of a live-attenuated Neethling virus to compare its genetic characteristics with LSDVs isolated from the clinical samples. Clinical samples including skin biopsies and scabs were collected in sterile cryovials using an aseptic technique, as has been described by the OIE [This study was undertaken in four districts of Chiang Mai Province, including Mae Wang, Doi Lo, Mae On, and San Pa Tong Districts, and two districts of Lamphun Province, namely Ban Thi and Ban Hong Districts . Skin no the OIE . Additio® Tissue kit , as described by the manufacturer’s instructions.Clinical samples of skin biopsies and scabs were sliced into small pieces at a weight of about 400 mg using a scalpel and homogenized in 500 μL of sterile 1X PBS solution at a pH of 7.4. Then, the tissue homogenates were processed for DNA extraction using a NucleoSpinCapripoxvirus was confirmed with the use of PCR by amplification of a 192 bp region in the P32 gene using a pair of primers: forward primer, 5′-TTTCCTGATTTTTCTTACTAT-3′, and reverse primer, 5′-AAATTATATACGTAAATAAC-3′, according to the PCR procedures, as described in a previous study [TM HS DyeMix , 21 μL of PCR water, 2.0 μL of extracted DNA, and 1.0 μL of each 10 μM primer concentration. Then, the PCR was constructed using a Bio-Rad S1000 ThermoCycler  under thermal conditions, including an initial denaturation at 94 °C for 5 min followed by 34 cycles of denaturation at 94 °C for 1 min, primer annealing at 50 °C for 30 s, a standard extension at 72 °C for 1 min, and a final extension step at 72 °C for 5 min. After that, the PCR products were separated in 1.5% Agarose gel and visualized under UV transilluminator for confirmation of LSDV-positive samples with a band size of 192 bp.The presence of us study . The PCRTM HS DyeMix , 21 μL of PCR water, 2.0 μL of the extracted DNA, and 1.0 μL of each 10 μM primer concentration. Then, the PCR was constructed under the following thermal conditions, including an initial denaturation step at 96 °C for 5 min followed by 35 cycles of a final denaturation at 95 °C for 30 s, an annealing at 50 °C for 30 s, an extension step at 72 °C for 30 s, and a final extension step at 72 °C for 5 min. After that, the PCR products were resolved on 1.5% agarose gel against a SibEnzymeTM 100 bp DNA ladder  at 100 V in 1X Tris-Acetic acid-EDTA (TAE) buffer containing 0.5 μg/mL RedSafeTM  for 30 min.All positive samples were processed to amplify the GPCR gene using PCR. PCR was performed using primers with the appropriate sequences (5′-TTAAGTAAAG CATAACTCCAACAAAAATG-3′ and 5′-TTTTTTTATTTTTTATCCAATGCTAATACT-3′) which were created for amplification of the entire GPCR gene at position 6961–8119 in the LSDV genome . Two addFollowing agarose gel electrophoresis on 1.5% agarose gel, the PCR amplicons were identified as the expected size using a molecular-weight marker. Then, the selected DNA bands were trimmed and purified using gel purification  according to the manufacturer’s instructions and transferred to Bio Basic  for DNA sequencing. After that, the quality of the obtained sequences was checked, and the ends of the sequences were trimmed using Molecular Evolutionary Genetics Analysis (MEGA) version X software . The similarity of the trimmed sequences was then tested with other GPCR gene sequences of LSDVs in GenBank using the National Center for Biotechnological Information’s (NCBI) web-based Basic Local Alignment Search Tool (BLAST). LSDV-specific signatures of these nucleotide sequences were also checked by translating the sequences to amino acid sequences followed by multiple sequence alignment using MUSCLE on the EMBL-EBI web server. After that, phylogenetic analysis was performed using MEGA X. LSDV sequences were selected after BLAST based on nucleotide similarity. Consequently, the origins of the isolates were representative of sequences from Asia, Europe, and Africa. Sequences were also selected from established LSDV vaccine strains, as well as those from the goatpox and the sheeppox viruses. Then, a phylogenetic tree was created using the Neighbor-Joining method in MEGA X, which used the evolutionary distances derived from identical branch lengths in the same sites to generate the phylogenetic tree . The maxSix suspected LSD outbreak areas in six districts of Chiang Mai and Lamphun Provinces were investigated in 2021. Suspected LSD cattle presented a range of clinical signs including depression, loss of appetite, fever, nasal and ocular discharges, enlarged superficial lymph nodes, circumscribed skin nodules on all parts of the body, and a decrease in body condition score .All of the farms in this study were dairy farms and included eight farms located in Chiang Mai Province  and four farms located in Lamphun Province (one farm from Ban Thi District and three farms from Ban Hong District).LSDVs were isolated from 26 dairy cows and 1 commercial vaccine were confirmed by PCR. Out of 26 dairy cattle, eight cows were dairy calves, with ages ranging from one to six months old. The rest included 17 lactating cows and one dry cow.Capripoxvirus family (Phylogenetic analysis clustered the northern Thai LSDV isolates, vaccine strains, and goatpox and sheeppox viruses into separate clades within the s family . There wThe present study investigated and confirmed LSD cases in northern Thailand using PCR. Additionally, LSDVs isolated from the identified outbreaks were characterized using GPCR gene sequencing. To the author’s knowledge, this is the first report on the molecular characterization and phylogenetic analysis of the LSDV that has circulated in this region in 2021.The first LSD outbreak in Thailand was discovered in the northeastern part of the country, in March of 2021. Subsequently, the disease was reported in the northern part of the country to include Chiang Mai and Lamphun Provinces in May of 2021. LSD-suspected cattle presenting LSD-like clinical signs were examined, and tissue samples were collected for LSD-infection confirmation using PCR. The results of PCR confirmed that LSD outbreaks occurred in this region. Chiang Mai and Lamphun Provinces are home to the highest density of dairy cattle when compared to other provinces in northern Thailand. Moreover, Chiang Mai Province has borders with a number of neighboring countries, and the incidences of cattle movement across these borders have been reported. These factors may present an increased risk for LSD outbreaks in this region . HoweverAfter GPCR gene sequencing, out of the 26 LSDV isolates, 24 isolates were grouped with LSDV isolates acquired from China, Hong Kong, and Vietnam. This finding suggests that the same LSDVs play an important role for outbreaks across shared borders. It has been reported that Thailand has a high risk of LSD outbreaks because live animal trade across borders from neighboring countries is quite high . MoreoveOut of 26 LSDV isolates, two isolates were grouped in vaccine strains after phylogenetic analysis. These LSDVs were isolated from cattle presenting LSD-like clinical signs within 7–14 days after vaccination. Remarkably, the cattle were healthy before vaccination and were acquired from LSD-free herds before outbreaks had occurred in northern Thailand. This finding suggests that the clinical signs of these cattle, such as circumscribed nodule skins, may have resulted as adverse effects of the LSD vaccine. A study in Jordan reported that certain adverse effects of the LSD vaccine occurred in animals within 1–20 days after being vaccinated. These effects included fever, nasal and ocular discharges, and variable-sized cutaneous nodules . HoweverThe LSDVs circulating in northern Thailand were found to contain the 12-nucleotide insertion in the GPCR gene. This result is similar to that of a study conducted in India and Bangladesh, which also found the 12-nucleotide insertion of the GPCR gene in LSDVs isolated from outbreaks in those countries ,22. FurtThis study firstly provides information on the molecular characterization and phylogenetic analysis of LSDV circulating in northern Thailand using GPCR gene sequencing. The results of this study indicate that the LSDV isolated from this region was genetically similar to the LSDVs circulating in China, Hong Kong, and Vietnam. This finding strongly supports the transboundary spreading of LSDVs throughout Asian countries. This information will be beneficial to researchers in better understanding LSDV molecular epidemiology and will contribute to the existing efforts of the government of Thailand in developing effective control strategies for LSDV.


Whey protein and sourdough ferment were used in different combinations to prepare functional sponge cakes, and their mutual influence on batter rheological behaviour as well as product physico–chemical, textural, colour and sensory properties were evaluated. All samples containing whey protein concentrate could bear the nutrition claim ‘a source of protein’. The substitution of wheat flour with whey protein significantly influenced batter viscoelastic behaviour, lowered cake-specific volume, increased product hardness, chewiness, gumminess, and browning index and modified its sensory characteristics. The incorporation of sourdough in protein-enriched sponge cakes improved product-specific volume and appearance compared to a protein-containing sample without sourdough. Although sourdough addition has less of a deteriorating effect on sponge cake rheological and textural properties, when combined with whey protein, it led to a significant reduction in batter elasticity and an increase in product hardness. It was also shown that spontaneously fermented sourdough cannot act as the only leavening agent in sponge cake production. In general, the results of this study have shown that sourdough addition can contribute to improvement in protein-enriched sponge cake quality and that further investigations are necessary in terms of different sourdough and flour type incorporation to minimize the negative effects of protein addition. Nowadays, food products undergo too many formulation and processing changes to fulfil modern world demands. These changes involve improving nutritional value, reducing, or fully replacing calorie-laden ingredients, and improving the taste and overall characteristics of the original product ,2. AlthoWithin the framework of improving the quality of food products, different studies have highlighted the necessity to apply some of the novel or modified food processing technologies. A trend that is increasingly attracting the attention of both the bakery industry as well as consumers is the application of sourdough technology as an approach for modification of flour functionality towards improvement in cereal-based products quality. Sourdough fermentation is a specific process where lactic acid bacteria and yeast create a complex consortium which uses the metabolism of both, as well as their mutual impact, to transform ingredients. Nutrients and bioactive compounds are thus subjected to numerous changes providing several benefits to final products in terms of nutritive, health, microbial and sensory aspects ,17,18,19Therefore, the aim of this study was to evaluate the effect of whey protein and spontaneously fermented whole wheat sourdough on sponge cake quality. Contrary to the other studies which monitored the effects of egg white protein replacement with whey protein concentrate on the physical, structural, and sensory properties of sponge cake, the present research gives an insight into changes that occur when wheat flour is substituted with whey protein concentrate. According to our knowledge application of sourdough fermentation and whey protein in sponge cake formulation is mostly examined separately and there is a lack of published data using sourdough technology to increase the quality of protein-enriched sponge cake. In order to determine the effects of combined approaches, several cake formulations were created. To estimate the quality of final products, rheological properties, chemical composition, physical properties, colour, textural analysis, as well as sensory evaluation were conducted.Wholemeal and refined wheat flours were obtained by Danubius d.o.o. . Whey protein concentrate  was purchased from Olimp Laboratories Sp.z.o.o. , while sucrose , eggs  and baking powder   were obtained from local market. Deionized water was used in all the experiments. Chemicals used in the analysis of sponge cakes were of analytical grade and were purchased from Merck .Wholewheat flour was used for creating sourdough starter. Spontaneously fermented sourdough was prepared through backslopping  under laboratory conditions .20), wheat flour and sourdough (S30) and wheat flour, sourdough, and whey protein at different ratios (S20P20 and S30P20). Furthermore, an additional sample with sourdough (S30P20BP0) was prepared using all ingredients, except baking powder. Mature and stable wholegrain wheat flour starter was used as sourdough in sponge cake formulations. In sourdough containing sponge cakes the amount of flour and water was reduced for the ones already present in added sourdough.Six sponge cake formulations were prepared according to Fresh eggs were separated into albumen (egg white) and yolk. Subsequently, 52.5 g of egg albumen was whipped with 20 g of sugar in a mixer  for 1 min at mixing speed 5. Moreover, an additional amount of sugar (65 g) and egg yolk (32.5 g) was also mixed for 1 min at speed 3 and added to previously prepared mixture. Subsequently, the appropriate amount of water was added and mixed for 1 min at speed 1. Finally, the powder ingredients  were homogenised and incorporated into previously prepared mixture by mixing for 1 min at speed 1. At the end, mature sourdough was added and gently mixed for 30 s. The obtained blends were poured into silicone moulds (60-mm diameter), 30 g each per sample. Afterwards, the poured batter was baked in a conventional oven at 200 °C for 11 min and 30 s. Two batches of each sample were prepared.Rheological properties of batter were examined, while physical characteristics, chemical composition, colour, textural properties, and sensory evaluation were determined on baked sponge cake.To assess the rheological properties of batter, a HAAKE MARS Rheometer  was used. Dynamic oscillatory measurements were performed using serrated parallel plate geometry measuring system  at constant temperature of 25 °C maintained by Peltier system. Mechanical spectra (frequency sweeps) were measured in the range 0.1–10 Hz at 1 Pa stress, which was within previously determined linear viscoelastic range. Three replicates were performed for each analysis.Methods of the Association Official of Analytical Chemists  were used to determine chemical composition of prepared sponge cakes. Moisture, fat, protein, ash and total fibre content were analysed . Total carbohydrates were calculated by difference following Equation (1) and total energy  was calculated according to Equation (2). All chemical analyses were performed in duplicates.3/g.The sponge cake samples volume was determined using laser measuring device VolScan Profiler 600  in triplicates. Laser step was set to 1 mm and rotation speed was set to 1 rps. Specific volume was calculated as the volume to mass ratio and expressed in cmThe colour of crust and cross-section of sponge cakes was determined by colourimeter Konica Minolta CR400  in five repetitions per batch. The obtained results were expressed in terms of L* (brightness/darkness), a* (redness/greenness) and b* (yellowness/blueness) according to CIELab system of colours. Browning index (BI) was calculated from equations  only forThe TA XT2 Texture Analyser  equipped with a 30-kg load cell was used to analyse texture of baked sponge cakes. Instrumental settings for TPA test (TPA.PRJ) were taken from the software package . Samples from the centre of the crumb slices were cut into cylinders  and compressed. Measurements were conducted in five repetitions per batch.Descriptive sensory analysis was performed with a panel of 10 well-trained sensory panellists  employed at the Institute of Food Technology, University of Novi Sad, Serbia. Training of panellists and descriptive terminology creation were performed on the four selected commercial sponge cakes. All panellists agreed on sensory attributes and definitions. The final list consisted of three appearance attributes , two odour attributes , three taste attributes , one flavour attribute (egg flavour) and four texture attributes  .The samples were cut in half and every assessor received one half served on plates labelled with 3-digit numbers in sequential monadic manner following a balanced presentation order. Sensory evaluation was performed in two separate sessions, in individual booths at room temperature; tap water was used for palate cleansing. The panellists recorded their perception of attribute intensities on a 10-centimetre continuous linear scale with left side of the scale corresponding to the lowest intensity and the right side corresponding to the highest intensity.The study was approved by the Ethics Committee of Institute of Food Technology in Novi Sad, University of Novi Sad, Serbia (Ref. No. 175/I/7-3 from 6 July of 2021).p < 0.05. Statistical analysis was performed by using software package XLSTAT 2022.1.2. Principal Component Analysis (PCA) was performed on the instrumentally measured textural, colour, rheological and nutritional properties and the sensory attributes scores of the sponge cake samples to differentiate them and to analyse possible relationships between them.Measurements were performed in appropriate number of repetitions and data were presented as average ± standard deviation and analysed for the statistical difference by using Analysis of variance (ANOVA) followed by the post-hoc Tukey’s HSD test at Since product formulation modification in terms of ingredients replacement or addition could affect a production process, batter rheology represents a valuable tool to determine and characterize product performance during different processing operations such as mixing, pouring or baking of batter. The batter for sponge cakes is a complex emulsion and foam system. Sponge cake is a baked product with a porous structure and is classified as a foam-type cake. The ingredients used in sponge cake formulation have a significant impact on the rheological properties of the batter and the texture of the sponge cake ,14. The As can be seen from 20 sample was characterized with similar G″ and G′ values (tan δ = 0.97). On the other hand, the samples with the addition of both ingredients expressed higher frequency dependence and pronounced viscous behaviour. Samples S20P20 and S30P20 were characterized with tan δ values of 1.08 and 1.21, respectively. Phase angle value further increased in the sample without baking powder (1.30) underling the significance of this leavening agent in cross-linked structure formation.To further determine the viscoelastic behaviour of the samples, the tangent of the phase angle (tan δ) was measured. The phase angle δ (tan δ = G″/G′) was calculated at defined frequency of 1 Hz. A tan δ < 1 value indicates a predominantly elastic behaviour, whereas tan δ > 1 indicates a predominantly viscous behaviour. The control sample expressed predominantly elastic behaviour with the lowest value of tan δ (0.51). Compared to the control, all other samples had higher tan δ values, which means that the addition of whey proteins and sourdough affected viscoelastic properties towards more viscous behaviour. Namely, the sample containing only sourdough (without whey proteins) had predominantly elastic behaviour with slightly higher values of tan δ (0.75) compared to the control. The PAlthough a decrease in batter elasticity with sourdough fermentation as a result of protein degradation and conformational changes due to proteolytic activity and environment acidity is already documented , the imp20 where only whey proteins were added, while the lowest protein content was detected in the sample without whey protein addition S30. According to Regulation (EC) No 1924/2006 of the European Parliament and of the council of 20 December 2006 on nutrition and health claims made on foods, samples with whey protein addition  could be claimed as a source of protein.The chemical composition of prepared sponge cake samples is shown in 20, S30 and S30P20 samples.In general, the addition of whey protein to a formulation led to progressive protein increase, and consequent carbohydrate decrease, which is in agreement with the studies performed by Alves et al.  and Cama30P20BP0).In the sponge cake systems, a limited amount of water is available, creating a competitive atmosphere in formulations with many different ingredients. The amount and composition of the aqueous phase are of high priority for batter density, where the great importance belongs to the surface-active components such as proteins which, together with other surface-active components, form films around trapped air bubbles . Taking 20P20 and S30P20) resulted in an appealing gas bubbles layout, bearing in mind that good distribution and presence of bubbles influence final product characteristics such as texture, colour and volume [30P20BP0 resulted in several large, fused bubbles which negatively affected the mentioned characteristics, as can be seen in As can be seen from the longitudinal and transversal section , the incd volume . On the p < 0.05) influenced the colour of sponge cake crust and cross-section (p < 0.05) darker crust surface and statistically (p < 0.05) lighter cross-section (except sample S30) compared to the control sample (Control). Similar results were reported by Sahagún et al. [p < 0.05) higher reddish tones  of cake crust and less greenish tones  of cake crumb than the control sample. No clear trend was observed for crust and crumb yellowness.The colour of the cake surface crust is one of the critical quality characteristics since it directly influences the initial consumer’s acceptance. Changes in sponge cake formulations significantly  indicates the purity of the brown colour. It brings valuable insights into processes in which enzymatic or non-enzymatic browning reactions take place. All enriched sponge cake samples were browner with higher values of browning index.p < 0.05) increased the intensities of all analysed textural properties in comparison to the control sample (Control).The textural properties of prepared sponge cake samples, presented as hardness, springiness, cohesiveness, gumminess, chewiness, and resilience, are given in 2 formed through the chemical reaction between leavening agents such as sodium bicarbonate and lactic acid influenced the expansion of the initially incorporated bubbles during mixing [30P20BP0 sample provides more resistance to compression leading not only to the increased hardness but also to the increased gumminess and chewiness. On the contrary, the S30P20BP0 sample showed the lowest springiness and cohesiveness. Namely, springiness is a textural parameter that indicates the ability of the sample to recover its height between two compression cycles in the TPA test. High springiness values are associated with a fresh, aerated product [p < 0.05) increased springiness of samples but only if leavening agent was added in formulation (P20 < S20P20 < S30P20 < S30 < Control). The sample containing whey protein and sourdough without a leavening agent (S30P20BP0) was significantly (p < 0.05) denser and had a lower ability to respond to deformation. Cohesiveness is a texture parameter that indicates the energy required for the second compression in the TPA test. This parameter can be correlated with the crumbliness, denseness and chewiness of products perceived by human senses [The observed correlation between the specific volume and cake hardness is similar to other studies , accordig mixing . The siz product . The addn senses .The results of the descriptive sensory analysis are presented in 2 production affects not only the texture but also the aroma of the final product. Furthermore, the incorporation of protein and sourdough caused an increase in sweet taste perception; however, only when 30% sourdough was added in formulations containing baking powder (S30 and S30P20) was the increase in noticed sweetness significant (p < 0.05). Accordingly, bitterness detected in the control sample was dramatically reduced in all other samples. These results may be the consequence of biosynthesis of the low-calorie sugar alcohols by lactic acid bacteria and yeasts in sourdough. Sahin et al. [All samples containing protein and/or sourdough created a more cohesive, denser, and less crumbly and adhesive structure compared with the control muffin. The less aerated structure of these products affected their odour and flavour. These samples showed a less pronounced overall odour, odour on eggs and flavour on eggs. According to Sahin et al. , lower Cn et al.  discusseThe absence of sodium bicarbonate in sponge cake formulation had a detrimental effect mostly on the textural properties producing a poorly aerated structure with a firm, dense, compact crumb structure that is less elastic. Similar results were observed with the instrumental measurements of textural properties. The main mechanisms that lead to foam destabilization are drainage-driven by gravity, coarsening due to pressure differences between bubbles of different sizes and coalescence, which is the bursting of liquid films separating neighbouring bubbles.30 and S30P20BP0 samples in different quadrants, while the separation of the other three samples was not so clear, which indicates that they are moderate in most of the analysed parameters. S30P20BP0 sample is separated mostly based on significantly higher values of instrumentally measured hardness, gumminess and chewiness, and high content of proteins. Contrary to these, the control sample is mostly separated due to highly pronounced crumbliness, intense overall odour, odour and flavour of eggs, and persistent intense sweet aftertaste evaluated by a sensory panel. Looking at the PC1–PC3 factorial plane, it can be observed similarities between the control and S30 in terms of high bitterness, sweetness, adhesiveness, pore uniformity and shape irregularity, high content of carbohydrates and low content of proteins. The obtained biplots emphasize the predominance of whey proteins in the determination of almost all quality parameters.Principal Component Analysis (PCA) was performed to explore the relationship between sensory attributes and instrumentally measured textural, colour, rheological and nutritional properties of sponge cake samples . PCA bipThe results of this study have shown that the replacement of flour with whey protein concentrate, as well as spontaneously fermented sourdough, led to the final product with specific quality characteristics. These ingredients significantly influenced batter viscoelastic properties, moving from predominantly elastic behaviour in the control sample to predominantly viscous behaviour in a combination of sourdough and whey protein concentrate. Both added components with their constituents, such as simple sugars and different types of proteins, resulted in darker crust colours (due to Maillard reactions) and changes in sensory  and textural properties (denser structure with lower specific volume). Despite the observed deteriorated textural properties, the overall characteristics of the final product were at a satisfactory level. In addition to the improvement in the nutritional quality of formulated sponge cakes, the results of this study suggest that sourdough addition can contribute to the improved volume and appearance of protein-enriched sponge cakes. Further research should be focused on the modification of sponge cake formulation with tested raw materials as well as the exploitation of the potential of other protein and sourdough sources in order to improve product techno-functionality, while preserving its nutritional potential.


Plasmodium falciparum parasites in the brain as well as multiple other organs. Thus, multiple organ dysfunction syndrome (MODS) may be present in pediatric patients with CM, but its frequency and association with mortality have not been evaluated. This is a retrospective study of data collected prospectively from children with CM admitted in Blantyre, Malawi. Physical examination findings and laboratory values necessary to calculate a Pediatric Logistic Organ Dysfunction–2 (PELOD-2) score, a validated method that quantifies organ dysfunction in critically ill children, were abstracted. A total of 145 patients were included. Mortality was 15% (n = 22). Ten patients (7%) had single organ dysfunction, 36 (25%) had two organs involved, 68 (47%) had dysfunction of three organs, and 31 (21%) patients had four organs affected. Beyond neurologic dysfunction, other organ systems involved included hematologic (77%), renal (61%), cardiovascular (44%), and respiratory (1%). The median PELOD-2 score on admission was 4 (interquartile range [IQR] = 3–6) in survivors and 6.5 (IQR = 5–10) in the nonsurvivors (P < 0.0001). Admission PELOD-2 score predicted mortality with an area under the curve of 0.75. MODS is widespread in pediatric patients with CM. Objectively identifying children with MODS, and therefore at an increased risk of mortality, may allow for the allocation of limited resources.Malaria resulted in an estimated 627,000 deaths in 2020, the majority of which occurred in children under 5 years of age. Cerebral malaria (CM) is a severe manifestation of the disease with case fatality rates of up to 40%. Autopsies in children with CM have demonstrated sequestration of  Plasmodium falciparum parasitemia, and no other discernible cause of coma, is one of the most severe manifestations of malaria. Despite advances in both malaria prevention and treatment, mortality rates in children with CM continue to range from 15% to 40%, with neurological sequalae identified in up to 50% of survivors at the time of long-term follow-up.–In 2020, malaria resulted in approximately 627,000 deaths worldwide with 96% occurring in Africa, primarily in children less than 5 years of age.P. falciparum parasitized red blood cells can be found adherent to the microvasculature of many organs within its host.–P. falciparum malaria have MODS when using the Sequential Organ Failure Assessment score to define organ dysfunction.,P. falciparum malaria had evidence of multiorgan involvement. Multiple organ dysfunction was present in 14 of the 17 patients (82%) with CM. In children with both CM and multiple organ dysfunction, case fatality rates were 14.3% compared with 5.9% in those with multiple organ dysfunction alone, but standard definitions of organ dysfunction were not used.In a study by Milner et al., autopsy results of 53 Malawian children with CM demonstrated significant sequestration of parasites within the brain, heart, lungs, spleen, stomach, small intestines, large intestines, and skin.The Pediatric Logistic Organ Dysfunction–2 (PELOD-2) score is a validated scoring system used to identify MODS and predict mortality in critically ill children .17 In thThis is a retrospective study of data collected prospectively from January 2019 to June 2021 at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi, as an ancillary study to Treating Brain Swelling in Pediatric Cerebral Malaria (5U01AI126610-02). The study was approved by the ethics committee at Michigan State University and at the University of Malawi College of Medicine Research Ethics Committee. All subjects’ guardians provided both verbal and written informed consent before enrollment.Plasmodium falciparum, and no other discernable cause of encephalopathy) were approached for enrollment. Demographic data, vital signs, physical examination findings, and laboratory studies were collected at the time of admission. The patient’s blood pressure was taken on a supported upper extremity at heart level when the child was at their calmest state, using an electronic sphygmomanometer . The correct cuff size was selected based on the circumference of the arm with the bladder length equivalent to 80% to 100% of the circumference of the arm and the width at least 40% to ensure the most accurate reading.The parents or caregivers of children 6 months to 12 years of age who met the WHO case definition of cerebral malaria . Venous blood was drawn to obtain a complete blood count, electrolyte analysis, and blood gas analysis . All patients received treatment of severe malaria according to the Malawi Standard Treatment Guidelines, which included the administration of intravenous artesunate, treatment of fever and hypoglycemia (defined as blood glucose < 2.2 mmol/L or 40 mg/dL), and the use of anticonvulsants and antibiotics if indicated. No patients were on inotropes, intubated, or ventilated at the time of admission.2/FiO2 ratio was used instead of the PaO2/FiO2 ratio as arterial blood gases are not routinely obtained as part of standard of care. A SpO2/FiO2 ratio of ≥ 100 was scored as 0 points and < 100 was scored as 2 points.,Paper charts of all eligible patients were reviewed. Data required for the calculation of each of the five subscores that comprise the PELOD-2 score were extracted for each patient. Criteria were met for organ dysfunction if the score was > 0 in any organ specific subscore. If more than one organ system had a score > 0, the patient was diagnosed with MODS. The total PELOD-2 score was calculated and recorded. For the neurologic subscore, BCS was used instead of the Glasgow Coma Score (GCS). A GCS was not obtained for any patient at the time of admission, but there was a documented BCS for each patient because it is more commonly used in children with CM. A BCS of 4 to 5 was scored as 0 points, 1 to 3 was scored as 1 point, and 0 was scored as 4 points.n (%) as appropriate. Comparisons between survivors and nonsurvivors were analyzed using the Fisher’s exact test for categorical data and Mann–Whitney test for continuous data. Correlation between age and PELOD-2 score was evaluated using the Spearman rank test. Receiver operator characteristic (ROC) curve analysis was used to assess the performance of the PELOD-2 score to predict mortality. Two-sided P values < 0.05 were considered statistically significant throughout. Analyses were performed using GraphPad Prism version 9.00 for Windows .Values are reported as median (interquartile range) or The 145 patients consecutively admitted between January 29, 2019, and June 2, 2021, were reviewed. Basic demographics, vital signs, and admission laboratories are illustrated in Thirty-six patients (25%) had two organs involved, 68 (47%) had dysfunction of three organs, and the remaining 31 (21%) patients had four organs affected. There was no significant difference between the number of organ systems involved in survivors and nonsurvivors . The difn = 22). The median PELOD-2 score on admission was 4 (interquartile range [IQR] = 3–6) in those who survived to discharge and 6.5 (IQR = 5–10) in the nonsurvivors . Ninety-five subjects had evidence of retinopathy. Presence of retinopathy was not associated with PELOD-2 score. For patients with retinopathy, median PELOD-2 score was 5 (IQR = 3–6) versus 5 (IQR = 3–6.25) for those who were retinopathy negative (P = 0.62). Retinopathy was not associated with mortality . Although age was associated with mortality in this cohort, age was not associated with PELOD-2 score . As a sensitivity analysis, we performed multivariable logistic regression with age and PELOD-2 score included in the model. When accounting for age, a higher PELOD-2 score remained associated with decreased odds of survival .Overall mortality for the cohort was 15%  in 135 patients (93%) at the time of admission. Neurologic dysfunction was present in all by definition. The hematologic system was the second most affected organ system, with dysfunction defined as leukopenia or thrombocytopenia. This finding is not surprising because thrombocytopenia is a well-recognized, common complication of malaria with several proposed underlying mechanisms including immune-mediated destruction, splenic sequestration, bone marrow suppression, and consumption in the microvasculature.,–,–,,–Renal dysfunction, defined as elevated creatinine level for age, was the third most common type of organ dysfunction in our cohort. Hypovolemia with resultant low renal blood flow and secondary renal ischemia, tubular necrosis due to hemoglobinuria and cytokine-induced apoptosis, and myoglobinuria from muscle necrosis are proposed mechanisms of acute renal failure in patients with severe malaria.,,Lastly, almost half of our cohort had cardiovascular dysfunction defined as elevated lactate levels, hypotension, or both. On the basis of prior studies, we believe the elevated lactate level (≥ 5 mmol/L) found in > 40% of our patients was likely multifactorial and a combination of the following: 1) poor perfusion with resultant tissue anoxia and subsequent anaerobic glycolysis, 2) decreased clearance of lactate in the setting of liver dysfunction, and 3) the release of lactate by parasitized erythrocytes.The presence of MODS was associated with increased mortality in our cohort of patients. Additionally, nonsurvivors were found to have higher PELOD-2 scores at the time of admission. There was no significant difference in the number of organ systems involved between survivors and nonsurvivors, suggesting that the association with outcome may be related to more severe involvement of the affected organs. The increase in mortality in pediatric CM patients in the presence of MODS illustrates the need for improved awareness of MODS so management strategies for these patients can include appropriate multiorgan, multisystem support. If a child with CM has cardiovascular dysfunction, maintaining appropriate cerebral perfusion pressure without pharmacologic assistance, particularly if intracranial hypertension is present, will be difficult.Our work has several limitations. This is a retrospective study that took place in a resource-limited setting. Although our center may have access to resources not widely available in other malaria-endemic regions, compared with higher income countries, healthcare resources are lacking. There were instances of missing data attributed to times when testing strips or laboratory reagents were not available. In these cases, scoring missing data as normal may have resulted in underestimating the overall frequency as well as the severity of MODS in multiple patients. We may have underestimated the presence of renal failure in our cohort because 15 patients (10%) were missing a creatinine at the time of admission. We propose the need for further studies investigating the frequency of renal failure in pediatric patients with severe malaria.2/FiO2 ratio. Despite other studies reporting the clinical recognition of respiratory distress in up to 40% of pediatric patients with severe malaria, only 1 out of 145 patients in our work met PELOD-2 criteria for respiratory dysfunction.Lastly, the potential limitations of the PELOD-2 scoring system itself must be considered when used in this population. For example, respiratory dysfunction in the PELOD-2 score is partially determined by the presence of mechanical ventilation, which is not a common intervention where malaria is endemic. Additionally, the ability to obtain an arterial blood gas is extremely rare in these settings, limiting the feasibility of calculating a PaOMultiple organ dysfunction is prevalent in pediatric patients with CM. MODS is associated with increased mortality in this population, and admission PELOD-2 scores may objectively identify children with CM at highest risk of mortality allowing for the allocation of limited resources. Given the frequency of MODS we identified, recognizing additional organ systems involved is essential when providing patient care and making management decisions.


Irregularities in breathing patterns can be detected using breath monitor sensors, and this help clinicians to predict health disorders ranging from sleep disorders to heart failures. Variations in humidity during the inhalation and exhalation of breath have been utilized as a marker to detect breath patterns, and graphene-based devices are the favored sensing media for relative humidity (RH). In general, most graphene-based RH sensors have been used to explore resistance change as a measurement parameter to calibrate against the RH value, and they are prone to noise interference. Here, we fabricated RH sensors using graphene ink as a sensing medium and printed them in the shape of interdigital electrodes on glossy paper using an office inkjet printer. Further, we investigated the capacitance change in the sensor for the RH changes in the range of 10–70%. It exhibited excellent sensitivity with 0.03 pF/% RH, good stability, and high intraday and interday repeatability, with relative standard deviations of 1.2% and 2.2%, respectively. Finally, the sensor was embedded into a face mask and interfaced with a microcontroller, and capacitance change was measured under three different breathing situations: normal breathing, deep breathing, and coughing. The result show that the dominant frequency for normal breath is 0.22 Hz, for deep breath, it is 0.11 Hz, and there was no significant dominant cough frequency due to persistent coughing and inconsistent patterns. Moreover, the sensor exhibited a short response and recovery time (<5 s) during inhalation and exhalation. Thus, the proposed paper-based RH sensor is promising wearable and disposable healthcare technology for clinical and home care health applications. Abnormalities in the human respiration rate is a vital sign of health disorders. For a healthy adult at rest, the normal breathing rate ranges from 12 to 20 breaths per minute. By detecting irregularities in the breathing rate and respiration pattern, clinicians can predict acute events such as chronic obstructive pulmonary disease (COPD) , chronicIn general, there are two methods for measuring the respiratory rate, i.e., contact-based and contactless ones . ContactIn recent years, paper has been considered as a promising sensor substrate owing to its low cost, flexibility, lightweight, biodegradability, and disposability ,14. GreaThe ideal humidity level that comforts the human body is between 40% and 70% at the appropriate temperature . The humAnother consideration is the sensor readout, which can be either resistive or capacitive. Many humidity sensors are resistive-type ones, which respond to a change in the resistance with relative humidity ,38. HoweAccordingly, we present an inexpensive, disposable, and sensitive capacitive-and paper based humidity sensor for respiration sensing. The sensor was fabricated through one-step fabrication technique, which only required an inkjet printer, glossy paper, and graphene printing ink. In addition, the paper-based humidity sensor was designed with an interdigitated electrode (IDE) pattern to increase the effective sensing area and the capacitance of the sensor. The RH-sensing characteristics of the fabricated sensor were investigated in the range of 10–70% RH. To demonstrate that the sensor enables continuous, real-time breathing monitoring, the sensor was studied under three different breathing situations  and the respiratory frequency rates and patterns were analyzed.Graphene printing ink with a viscosity in the range of 10–20 cP was purchased from GO Advanced Sdn. Bhd. . A4 glossy inkjet photo paper (180 gsm) and dual-sided conductive copper tape (5 mm × 20 m) were obtained from INRO Electronic . The Arduino Mega2560 and BME680 environmental sensors were purchased from Cytron Sdn. Bhd. .−1 range using Raman Spectroscopy (Renishaw InVia Raman Microscope). The surface contact angles for a 20 μL droplet on the surfaces of glossy paper with printed graphene and without printed graphene were measured using a goniometer .All the paper sensors were prepared using Brother Model MFC-J200 inkjet printer. The surface morphology of graphene-printed glossy paper was characterized using a field emission scanning electron microscope (FESEM) . The chemical composition of the graphene-printed surface was characterized by X-ray photoelectron spectroscopy (XPS) . Raman spectra characterization with a 532 nm excitation was performed in the 3200–100 cmThe device fabrication protocol for the paper-based Inkjet-printed-graphene sensor is schematically shown in 3 with two peripheral holes that acted as the inlet and outlet. The inlet and outlet were always open for the continuous flow of gas mixture through the chamber to achieve a constant gas pressure. Purified nitrogen, N2, gas was supplied by a gas tank  at a pressure of 1.5 bar was regulated by both of the MFCs . During humidification, the N2 gas was flowed through a bubbler filled with deionized water into the sensing chamber at a fixed flow rate of 200 sccm, which was regulated by MFC2. The chamber was dehumidified by flowing dry N2 gas through the chamber at flow rate 711 sccm, which was regulated by MFC1. The fabricated paper sensor was placed in a homemade sensing chamber that was interfaced with the microcontroller for the humidity detection tests. Further, a data logging system was utilized to record the RH conditions using a PC. The operating ranges of the chamber for RH were controlled and monitored to stay within the range from 10% to 70%. The sensitivity of the sensor is expressed as Equation (1), where ΔC is the capacitance change due to the humidity, and ΔRH is the relative humidity variation [The RH sensing setup is illustrated in ariation .(1)S=[lThe description of the experimental setup used to investigate the response of the printed paper sensor to variable breath conditions is as follows: The paper sensor was embedded into a commercial KN94 mask for human respiration monitoring, as shown in FESEM was applied to examine the morphology of the printed sensing material at varied magnifications. The SEM images of the bare glossy paper (i–iii) and the graphene-printed glossy paper (iv–vi) at magnifications of 10 K, 20 K, and 50 K are shown in Hydrophilicity was investigated by measuring the water drop contact angle of the bare paper substrate and graphene ink film samples. 3 carbon and aromatic sp2 carbon are within the range of 284.5–285 eV [−) could be due to the water vapor on the graphene-printed paper surface and its origin in the surrounding environment [−1) and broad G-band (1584 cm−1), indicating the graphene layer that was inkjet printed onto the paper substrate [2 bonded carbon atoms in the graphitic structure [2 crystal structure due to oxidation [In addition, the elemental composition analysis on the energy dispersive XPS chart of the graphene-printed paper surface further confirms this result, illustrating the expected elemental peaks of carbon (C1s) and oxygen (O1s). From the XPS survey spectra in 5–285 eV ,45. The ironment . In Figuubstrate . The pretructure . The prexidation ,50.p value is 0.247 , which indicates that a not statistically significant difference occurred from day 1 to day 10. As a result, paper sensors can maintain good short-term stability during humidity testing. The repeatability of test on the paper sensor was evaluated by performing three cycles within the intraday (within same day) and interday (between days) range from 10% to 70% RH. The results of the paper sensor are considered to be highly repeatable, with relative standard deviations (RSD) of 1.2%  in terms of the capacitance (pF) value and the breathing pattern, with fast response and recovery times of 2.46 s ± 0.34 and 3.86 s ± 0.56 for the exhalation rise time, and 2.63 s ± 0.49 and 4.27 s ± 1.02 for the inhalation fall time under normal breathing and deep breathing conditions, respectively. The prototype sensor has a low cost, and it is disposable when it is used for frequent breathing pattern recognition. The sensor requires the simple inkjet printing of graphene ink on glossy paper using a typical office inkjet printer. The method is also suitable for mass production at a low cost. However, there are limitations in terms of aligning the layer features while one is printing multiple layers on same piece of paper. Furthermore, functionalized graphene ink for the selectively sensing of volatile organic compound molecules in breathe can be investigated in future studies.


Depressive disorders are the leading contributor to medical disability, yet only 22% of depressed patients receive adequate treatment in a given year. Response to treatment varies widely among individuals with depression, and poor response to one treatment does not signal poor response to others. In fact, half of patients who do not recover from a first-line psychotherapy will recover from a second option. Attempts to personalize psychotherapy to patient characteristics have produced better outcomes than usual care, but research on personalized psychotherapy is still in its infancy. The present study explores a new method for personalizing psychotherapy for depression through simulation modeling. In this study, we developed a system dynamics simulation model of depression based on one of the major mechanisms of depression in the literature and investigated the trend of depressive symptoms under different conditions and treatments. Our simulation outputs show the importance of individualized services with appropriate timing, and reveal a new method for personalizing psychotherapy to heterogeneous individuals. Future research is needed to expand the model to include additional mechanisms of depression. Depressive disorders are the leading contributor to medical disability , yet onlWhile attempts to personalize psychotherapy to patients have produced better outcomes than usual care ,7,8, resDepression is a highly complex condition that involves a large number of interacting mechanisms, thereby requiring a method capable of representing multiple non-linear interactions. One method for understanding complex problems and the impact of potential interventions is system dynamics (SD) simulation method. SD is a method that has been used to identify ways to manage and prevent chronic diseases, mostly at the population-level, and for public health more generally ,13. SD mA central element to SD modeling is to identify and operationalize ’feedback loops’. Feedback loops are particularly important because they regulate dynamic phenomena  and can The present study aims to use the SD model of depression, rumination, and stress to explore a method for personalizing psychotherapy. Specifically, this study will examine whether mindfulness-based cognitive therapy (MBCT) has a different effect on adolescents who differ by prior life stressful events, ongoing stressors, rumination, and trajectories of depressive symptoms. Since people often delay seeking treatment for up to eight years after experiencing a first episode of depression , this stThis study was declared exempt by the Michigan State University Institutional Review Board.n = 520 female; n = 545 male) from grades 6 to 8 in two middle schools in central Connecticut. The sample included 56.9% (n = 610) Hispanic/Latino, 13.2% (n = 141) non-Hispanic White, 11.8% (n = 126) non-Hispanic Black, 9.3% (n = 100) biracial/multiracial, 2.2% (n = 24) Asian/Pacific Islander, 0.8% (n = 9) Middle Eastern, 0.2% (n = 2) Native American, and 4.2% (n = 45) other racial/ethnic groups. Based on school records, 62.3% of students were eligible for free or reduced lunch. The socioeconomic status of the community in which the two schools are located is rated low because the income per capita is $18,404.Participants were 1,065 adolescents  were contacted to provide active consent. Parents who did not return the consent form were then contacted by phone. About 22% of parents could not be reached by phone, and six parents refused to let their child participate in the study. Adolescent participants provided written assent. The participation rate at baseline was 72%. Two additional assessments were conducted after the first assessment. The first and second assessments were four months apart, and the second and third assessments were three months apart. Of those who completed the first assessment (T1), 28% (n = 221) did not complete the second assessment (T2), and 20.4% (n = 217) did not complete the third assessment (T3). The primary reason for attrition was due to students leaving the school district. Those who did not complete the second and third assessments were more likely to be female (χ2 (1) = 6.85, p < 0.01), however, they were not different in terms of grade level, race/ethnicity, or being from a single-parent household (ps > 0.10). Depressive symptoms and rumination level of those who did not complete at least one of the follow-up assessments did not differ from those who completed all assessments (ps > 0.10). Depressive symptoms and stressful life events were collected at T1 and T3 while the level of rumination was measured at all assessment times .This study used existing data collected by McLaughlin and Nolen-Hoeksema . A full The Life Events Scale for Children  includesalmost never = 1, sometimes = 2, often = 3, or almost always = 4). Sample items include “Think about how alone you feel,” “Think about a recent situation wishing it had gone better,” and “Think why can’t I handle things better?” The CRSQ-Rumination exhibited good reliability in this sample (α = 0.86) and in previous research [The Children’s Response Style Questionnaire (CRSQ)  is comporesearch .α = 0.82).The Children’s Depression Inventory (CDI) is a 27 item measure of depressive symptoms in children and adolescents . Each itOur SD simulation model of depression simultaneously captured the reciprocal relationships among depressive symptoms, rumination, and stressors at the individual level. SD models are often used to understand complex systems and the endogenous feedback mechanisms underlying observed trends . This meUsing SD models to study depression provides three advantages. First, SD models can capture multiple feedback mechanisms, latent variables, and accumulations. Although simultaneous equation methods and structural equation models can include feedback and latent variables, SD models incorporate all of these and also allow for nonlinearities. Second, SD models provide insights about the behavior of the system by showing the simulation results over continuous time. Third, SD models incorporate broad and realistic feedback mechanisms that allow for designing and analyzing different interventions for the same simulated individual.rumination causes more depressive symptoms and more depressive symptoms leads to even more rumination [past stressor kept alive increases, the outflow let it go elevates which leads to lower past stressors kept alive.The SD model that we developed in this study included two reinforcing feedback loops . It is bmination . The balPast stressors kept alive and depressive symptoms influence rumination with a delay. Thus, current rumination is the first order delay (weighted average) of indicated rumination. Indicated rumination is a linear function of past stressors kept alive [depressive symptoms [gender [The formula of the model is as follows. pt alive , depresssymptoms , and gen [gender .Rumination does not influence depressive symptoms immediately, as a result, depressive symptoms is formulated as the first order delay of indicated depressive symptoms that we assume is a function of rumination.indicated rumination and indicated depressive symptoms are not deterministic and are influenced by random events outside the model boundaries such as unmeasured contextual influences. Usually, those events are serially correlated, as a result, we added first-order auto-correlated noise terms that are normally distributed to the indicated rumination and indicated depressive symptoms. In turn, θ10 and θ11 are the standard deviation of these noise terms and θ12 is the correlation time.In addition, past stressors kept alive contains the memories of stressors not the stressors themselves  and outflow .The stock of 1-θ12 in All formulas are listed in n = 661) as well as separately for boys (n = 308) and girls (n = 353) are listed in p = 0.03) and Time 3 (p = 0.08) and higher levels of rumination at all evaluation times (p = 0.00). There is no gender difference in stressful life events at Time 1 (p = 0.98) and Time 3 (p = 0.27).Means and standard deviations of all measures at each evaluation time for all participants (1-θ12) are the same for all individuals, the model generates different trajectories for participants because the initial values of depressive symptoms, rumination, gender, prior stressors , and ongoing stressors  differ across individuals. A simulated individual with sizable ‘prior stressors’ has experienced many stressors in the six months prior to the beginning of the simulation and a simulated person with a large value of ‘ongoing stressors’ represents someone who experiences stressors during the entire simulation.Although the parameters of the model  and no more stressors occur in the lives of participants in group 2. Depressive symptoms of group 2 reach a state of equilibrium after around 10 years because it takes a long time for group 2 to fully let go of the stressors. Girls in group 1, unlike group 2, experience stressors during the entire simulation. As a result, stressors accumulate in the stock of past stressors kept alive and rumination and depression reinforce each other and increase over time (both reinforcing loops R1 and R2 dominate the dynamics of the system). Prior stressors in combination with initial rumination influence the trajectory of depressive symptoms in the first few months. Depressive symptoms initially grow if both prior stressors and initial rumination are high because high initial rumination reduces let it go rates, thus, prior stressors stay longer in the stock of past stressors kept alive and rumination and depression intensify each other (R1 and R2 dominate). However, their impact is temporary and if there are no ongoing stressors, depressive symptoms decline and become stable over time (B1 dominates). For instance, group 10, which is high in depressive symptoms and ongoing stressors, experiences an initial increase in depressive symptoms while the depressive symptoms of group 12 or 14 only increase slightly to reach the steady-state level in the absence of any ongoing stressors. However, the interaction of initial rumination and prior stressors has a long-term impact if large levels of the two factors are accompanied by large ongoing stressors (groups 1 and 9). Group 9’s depressive symptoms are initially low but elevated initial rumination combined with large prior and ongoing stressors increase their symptoms to a level similar to Group 1 (Reinforcing loops R1 and R2 dominate the system).The major impact of ongoing stressors on depressive symptoms can be observed by comparing column 1 with 2 and column 3 with 4 in The average and 75% envelope of the simulated depressive symptoms for 16 male adolescent groups are shown in Next, we will describe results from our test of the effects of MBCT on the depressive symptoms of heterogeneous adolescents in a simulated environment. We developed a test based on the empirical literature in which a stressful life event with an intensity of 40 was applied at month 20 and continued for two months; MBCT was then received at month 30. Prior findings have shown that the impact of therapy may last up to 24 months . SimilarThe dashed line in Comparison of columns 1 and 3 with 2 and 4 of the simulation results shows that those with ongoing stressors benefited the most from therapy. For instance, individuals in group 1 treated with MBCT after the intense stressful event Click here for additional data file.S2 Fig0, R0, S0, and SI represent initial depressive symptoms and rumination, prior stressors, and ongoing stressors respectively.D(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.