Although previous research has linked polypharmacy to lower cognitive function in the general population, we know little about this association among economically challenged African American (AA) older adults. This study explored the link between polypharmacy and memory function among AA older adults. This community-based study recruited 399 AA older adults who were 65+ years old and living in economically disadvantaged areas of South Los Angeles. Polypharmacy (taking 5+ medications) was the independent variable, memory function was the outcome variable (continuous variable), and gender, age, living arrangement, socioeconomic status , health behaviors (current smoking and any binge drinking), and multimorbidity (number of chronic diseases) were the covariates. Linear regression was used for data analyses. Polypharmacy was associated with lower scores on memory function, above and beyond covariates. Among AA older adults, polypharmacy may be linked to worse cognitive function. Future research should test the mechanisms by which polypharmacy is associated with lower levels of cognitive decline. There is a need for screening for memory problems in AA older adults who are exposed to polypharmacy. Polypharmacy (P), commonly defined as taking 5+ medications a day , is commPolypharmacy increases the risk of a wide range of undesirable health outcomes and events such as adverse drug events and drug–drug interactions ,5. PolypAlthough polypharmacy is more common in older adults ,21, veryAlthough polypharmacy is associated with cognitive decline and memory loss ,15,25,26Memory loss is a major problem in older adults ,28, inclThe main aim of this study was to investigate the association between polypharmacy (taking 5+ medications)  and memoThe current cross-sectional study, which was conducted in South Los Angeles, CA, USA, was composed of two sections: (1) a self-reported survey (interview) that collected data on demographics, SES, physical health, and mental health, and (2) a comprehensive evaluation of medications taken.The first part of this study was composed of a survey that took about 60 min to answer and was made up of the multi-item responses to health questionnaires on various topics such as chronic disease, substance use, mental health, pain, sleep, exercise, diet, quality of life, etc. The second part of this study included a comprehensive evaluation of all medications that were being used by the participants. This went beyond taking a simple medication history as all medications and their dosages were recorded by a physician or a nurse. This part of the data collection was not self-reported, and medications were provided by the patients. These included both PRN (As Needed) and scheduled medications and included both over the counter (OTC) and prescribed (RX). Given the nature of the data collection, we defined our medication data collection as comprehensive. Data were gathered between 2015 and 2018 ,41,42,43The participants needed to answer the survey items themselves. No help from a translator or a caregiver was acceptable. In addition, participants needed to conduct a full interview in English. This was decided because our participants were all AAs, defined as Blacks who were born and raised in the US. Thus, no other language was provided.The ethical aspects of this study received approval by the Charles R. Drew University (CDU) Institutional Review Board (IRB number = 14-12-2450-05). All the participants provided signed informed consent at the time of enrollment in the study. Participants also received some financial compensation. As mentioned above, the data were collected in two sections: (1) a structured face–to–face interview for evaluation of health, and (2) a comprehensive investigation of all medications that were being taken by the participant. Our study used consecutive, non-random sampling to enroll participants. Participants were enrolled if they were (1) AA, (2) older adults defined as 65 years or older, (3) living in economically challenged areas in South Los Angeles, (4) could conduct a full health interview in English. Exclusion criteria included (1) institutionalized individuals, (2) enrollment in other clinical trials . This approach increased the external validity of our results to the general population. The current analysis included 399 AA older adults (age ≥ 65 years).Participants were recruited from 16 predominantly AA churches, 11 senior housing apartment units, and some low-income public housing projects located in Service Planning Area (SPA) 6, LA County. Participation of the individuals was facilitated and encouraged by church leaders and housing apartment managers. Given that SPA6 is among the most economically disadvantaged areas, we were able to recruit community-dwelling members that were historically underserved and low income. The vast majority of older adults in SPA6 are AA (49%). From about 10.3 million individuals who live in LA County, 1.3 million are older adults. As LA County is the most populous county in the US, and given its large size (4300 square miles), LA County is divided into eight Service Planning Areas (SPAs). These SPAs have a more homogenous economic and demographic composition. These distinct regions facilitate surveillance and provision of public health and clinical services in a more tailored manner. Approximately 28–36% of SPA 6 households are below the federal poverty level and are uninsured. In SPA 6, about 60% of adults have income levels less than 200% of the federal poverty line (FPL), which is 1.5 times more than LA County overall. As we drew participants from SPA 6, participants were economically challenged. Across various SPAs, SPA 6 has the highest percentage of low-income AA and Hispanic people. Overall, SPA 6 is the most economically disadvantaged area of LA County. SPA 6 has recently experienced a rapid increase in the percentage of homeless AA individuals. Although all of LA County is experiencing an increase in homelessness, this is a more severe challenge in the poorest areas where AA individuals are more likely to lose their homes due to an inability to pay mortgage, rent, or property tax. Given poverty is concentrated in SPA6, homelessness has increased in this section of LA County . The study variables included age, gender, SES, multimorbidity, polypharmacy, and memory function. Demographic covariates. Gender, age, and living arrangement were the demographic variables. Gender  and living arrangement  were binary variables. Age was a continuous variable.Socioeconomic status. The two SES indicators in this study were educational attainment and financial strain. Educational attainment (years of schooling) was a continuous variable. To measure education attainment, we counted all years of schooling, including not only primary but also higher education (college years). Financial strain was measured using three items that measure whether participants lack enough money for what is essential to meet primary needs, namely (1) food, (2) clothes, and (3) utility bills. Responses for each item were on a 5-point Likert scale . We built a sum score with a range between 3 and 15. A high score reflected more financial strain (lower SES). Cronbach’s alpha of this study was 0.923  between polypharmacy and multimorbidity was 0.35). For multivariable analysis, a linear regression model was used. In our multivariable models, polypharmacy was the predictor variable, memory function was the dependent variable, and demographics, SES, and health were the covariates. We used the Enter selection so variables remained in the model even when they were not statistically significant. Beta (regression coefficient), standard errors (SE), 95% CI, and polypharmacy values were reported. p values equal to or smaller than 0.05 were considered significant. To perform data analysis, we used SPSS 23.0 . To describe the participants, we reported mean, standard deviation (SD), and frequency , depending on the variable type. For bivariate analysis, we used the Spearman correlation test. This test helped us test for any possible collinearity between the study variables. As Table 2 shows, there was no collinearity between any variables  or women . Overall, 75.4% of the participants were implementing polypharmacy (5+ medications per day). On average, participants were taking 7.6 (SD = 4) medications, with a range from 2 to 22. Memory function showed a range from 1 to 80 with a mean score of 52.11 (SD = 12.50). This study revealed an association between polypharmacy and lower memory function in economically disadvantaged AA older adults, above and beyond SES and health confounders that included age, gender, SES, and health (multimorbidity). The finding that low-income AA older adults with polypharmacy are at an increased risk of memory loss is in line with the literature that suggests polypharmacy may be one of the contributing factors to cognitive and memory decline . In a stResearch suggests that clinicians and practitioners who work with economically disadvantaged AA older adults should know about the link between polypharmacy and memory impairment in their population. Although more research is needed, inappropriate polypharmacy should be screened and prevented in all low SES AA older adults, particularly those with memory loss. Thus, the results of this study can help physicians/prescribers to reduce their likelihood of prescribing inappropriate polypharmacy. A side observation was that more than half of the sample population were living alone, considering the AA community is associated with a collective instead of an individualistic culture. We also observed a mean of 12.8 years of education, which suggests that our sample was a well-educated group. Participants were still living in low SES areas, suggesting that education has smaller than expected effects on changing AA life conditions ,54,55,56Our study is with some limitations. First, this was a cross-sectional study. Such a design rules out any causal inferences. Thus, our result on the link between polypharmacy and memory loss should be interpreted as an association rather than causation. Other limitations included low sample size and non-random sampling. Another limitation was reliance on self-reported data on multimorbidity. As mentioned before, we solely relied on self-reporting to measure CDs. Although some biases are possible, research has shown the high validity of self-reported CDs . Future There are high-risk and low-risk medications for memory and cognition decline. High-risk medications include benzodiazepines and anticholinergic agents. Future research should test the specific types of medications that are linked to more severe memory impairment. Similarly, there is a need to study domains of cognition other than memory function. In our study, we only had the number of medications, thus further analysis should also focus on the type of medications. Future research should also study other confounders within and beyond medications and chronic diseases.In summary, polypharmacy is associated with memory loss in economically disadvantaged AA older adults. Interventions that wish to address cognitive decline in particular and memory loss, in particular among AA older adults, may benefit from addressing medication-related challenges such as polypharmacy. There is a need for interventions that test the effects of programs on cognitive function in AA older adults. Future research is needed to understand whether drug–drug interaction is responsible for the link between polypharmacy and various aspects of cognitive decline beyond memory loss, and if we can undo this link for AA older adults.


BCR-ABL1 negative (aCML) is a rare myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with a high rate of transformation to acute myeloid leukemia, and poor survival. Until now, the diagnosis has been based on morphological grounds only, possibly making the real frequency of the disease underestimated. Only recently, new insights in the molecular biology of MDS/MPN syndromes have deepened our knowledge of aCML, enabling us to have a better molecular profile of the disease. The knowledge gleaned from next generation sequencing has complemented morphologic and laboratory WHO criteria for myeloid neoplasms and can provide greater specificity in distinguishing aCML from alternative MDS/MPN or MPNs. The most commonly mutated genes (>20%) in aCML are SETBP1, ASXL1, N/K-RAS, SRSF2, and TET2, and less frequently (< 10%) CBL, CSFR3, JAK2, EZH2, and ETNK1. Several of these mutations affect the JAK-STAT, MAPK, and ROCK signaling pathways, which are targetable by inhibitors that are already in clinical use and may lead to a personalized treatment of aCML patients unfit for allogeneic transplant, which is currently the only curative option for fit patients. In this review, we present two emblematic clinical cases and address the new molecular findings in aCML and the available treatment options.Atypical chronic myeloid leukemia,  BCR-ABL1 negative myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with a high rate of transformation to acute myeloid leukemia (AML), and is historically characterized by poor survival. The challenges of aCML management affect both diagnosis, due to the heterogeneity of the disease clinical features and the absence of unique biomarkers, and treatment choices, since no current standards of care exist.Atypical chronic myeloid leukemia (aCML) is a rare Here we describe two representative aCML clinical cases presenting in two different age groups and review the clinicopathologic features and management of the disease. The clinical characteristics and outcome of the two patients are summarized in 9/L with absolute neutrophil count (ANC) 34.2 × 109/L, hemoglobin (Hb) was normal and platelets (Plts) were 103 × 109/L. Manual differential revealed 46% hypogranulated and hypolobated neutrophils, 12% band forms, 12% metamyelocytes, 6% monocytes, 6% myelocytes, 2% promyelocytes, 2% eosinophils, and 14% lymphocytes. No hepatosplenomegaly was noted on physical examination. A bone marrow (BM) examination and trephine biopsy were performed and revealed hypercellularity with a significant myeloid expansion without excess of blasts and with mild dysplasia. Dyserythropoiesis and dysplastic megakaryocytes, including hypolobated forms, were noted  were 41.5 × 10re noted . KaryotyTET2  and one pathogenic mutation in EZH2 . After confirming the diagnosis of aCML, the ETNK1 gene was sequenced by Sanger method and resulted wild type.As a next step, the diagnostic sample was subjected to next generation sequencing (NGS) analysis using TruSight Myeloid Sequencing Panel (Illumina), covering 54 genes mutated frequently in myeloid malignancies see , which r9/L, ANC 14.7 × 109/L). He had a previous history of transitional cell carcinoma treated with radical cystectomy almost three years before referral and in complete remission ever since. In his medical history, he had a first hematology referral due to erythrocytosis at the time of the bladder neoplasia. At that time, a complete, first level molecular biology assessment for Philadelphia negative (Ph-) MPN was carried out and resulted negative, leading to the conclusion of a spurious erythrocytosis. An automated differential showed 90% neutrophils. The manual differential revealed 30% neutrophils with Pelger-Huet nuclear abnormality, 17% band forms, 6% metamyelocytes, 8% monocytes, 15% myelocytes, 14% promyelocytes, 1% blasts, and 9% lymphocytes. Fluorescence in situ hybridization (FISH) for t and molecular assessment for BCR/ABL1, JAK2V617F, CARL, and MPL were tested again at this time, and all resulted negative. The bone marrow (BM) aspirate was hypercellular without increased blasts but with subtle dyserythropoiesis and dysmegakaryopoiesis primarily consisting of hypolobated megakaryocytes with separate nuclear lobes , SRSF2 , and TET2 . A new mutation (p.H243P) in one of the hotspots described by Gambacorti-Passerini et al. [A 71 year old man was referred to our Hematology department due to leukocytosis , and the leukocyte alkaline phosphatase level may be low, normal, or increased, therefore lacking diagnostic utility [The clinico-pathologic characteristics of aCML include splenomegaly and a neutrophilic leukocytosis with left shift and prominent granulocytic dysplasia  . The WBCe marrow . Due to  utility ,5. A hyp utility ,7.9/L) at presentation, increased immature precursors in the PB, female gender, and age > 65 years [ASXL1, SETBP1, and TET2 genes have been associated with a more aggressive disease [The median overall survival (OS) of aCML patients is between 12 and 25 months ,10,11,1265 years ,10,13. I disease .The risk of progression to sAML seems to be higher in case of palpable hepato- or splenomegaly, monocytosis, BM blastosis > 5%, marked dyserythropoiesis, and transfusional requirement .Atypical CML is a challenging myeloid malignancy with features of both myeloproliferative and myelodysplastic syndromes. The MDS/MPN category was introduced in the 2011 WHO classification to include myeloid neoplasms with clinical, laboratory, and morphologic features that overlap MDS and MPN ,4. In adCurrently, the diagnosis of aCML is reached primarily on morphology-based criteria, as its clinical and molecular features overlap with those of other myeloid malignancies. No molecular abnormality detected to date is specific of aCML, and multiple mutations are often present in various combinations, due to the malignant multi-step clonal evolution of myeloid malignancies ,16.9/L with ≥ 10% of immature granulocytes, and ≤ 20% blasts in the blood and the BM [The main feature characterizing aCML is the presence of neutrophilic leukocytosis and marked dysgranulopoiesis. Moreover, to fulfil the diagnostic criteria, WBC should be ≥ 13 × 109/L), fewer immature granulocytes (< 10%), no granulocyte dysplasia, and no excess of blasts.These findings can distinguish aCML from CNL, that presents with higher WBC count , PDGFRB (5q31–33), FGFR1 (8p11), or JAK2 (9p24).The diagnostic work up should start with complete blood count with manual differential, morphological examination of PB smear, BM examination with assessment of dysplastic features, cytogenetic analysis and FISH to exclude Ph chromosome and rearrangements involving Once CML has been excluded, second level testing should include human leukocyte antigen (HLA) typing in transplant eligible patients, and assessment with a NGS myeloid panel, not only to confirm diagnosis but also to open the possibility of a target therapy.An algorithm summarizing the diagnostic work-up is reported in The molecular features of aCML include an increased frequency of gene fusions or an aneuploid karyotype. One or two chromosomal aberrations, e.g., trisomy 8 or 9, del 20q), −7/7q or isochromosomes 17q, are found in up to 50% of patients 0q, −7/7q but are Although in many myeloid malignancies there is prognostic significance associated with abnormalities of chromosome 7 and complex cytogenetics, no study to date has reported their prognostic role in aCML or in other MDS/MPN, with the exclusion of CMML.The heterogeneity of aCML, both clinical and molecular, is partly due to the difficulty in comparing reports of this rare disease in case series characterized over several decades, during which diagnostic criteria were redefined and novel DNA sequencing techniques emerged.The knowledge gleaned from NGS has complemented morphologic and laboratory WHO criteria for myeloid neoplasms and can often provide greater specificity in distinguishing aCML from alternative MDS/MPN or MPNs . HoweverASXL1 and TET2, a frequent finding in aCML, correlate with older age and monocytosis (TET2), are related to “clonal hematopoiesis of indeterminate potential” (CHIP), and are present in the founder clones of both MDS and MPN. [TET2 mutations [The mutational landscape of MDS/MPN represents an overlap between those of MDS and MPN. Similar to MDS, the majority of cases of MDS/MPN harbors at least two mutations involving epigenetic regulation and splicing factors in the dominant clone, with acquisition of additional mutations over time . Moreoveutations .SETBP1, ASXL1, N/K-RAS, SRSF2, and TET2, and less frequently (< 10%) CBL, CSFR3, JAK2, EZH2, and ETNK1 [JAK2, CALR, and MPL genes are wild type, although a few cases of CMML and aCML have been reported to harbor JAK2 mutations [The most commonly affected genes > 20%) in aCML are 0% in aCMnd ETNK1 ,22,23,24utations ,25.SETBP1 and ETNK1 appear to be the alterations most closely associated with aCML, even though they are not univocally disease-specific [SETBP1 and ETNK1 may be useful diagnostic tools for the disease [Mutations in specific ,21,22,26 disease ,21,22,26SETBP1 have been identified in up to one third of aCML patients as compared to 10% only of MDS/MPN-U and 6–15% of CMML, and occasionally in JMML or AML secondary to MPN or MDS [SETBP1 are associated with a more adverse clinical presentation , suggesting that this alteration is not only relevant to leukemic oncogenesis, but also provides important prognostic value [Recurrent mutations of N or MDS . Multiplic value .ETNK1 were identified at a prevalence of 8.8% in sixty-eight cases of aCML. All mutations were heterozygous, and in two cases occurred concomitantly with SETBP1 mutations. ETNK1 encodes an ethanolamine kinase, catalyzing the first step of the phosphatidylethanolamine biosynthesis pathway, which is critical for regulating the cell membrane architecture and the topology of transmembrane domains of membrane binding proteins [ETNK1 mutations is not completely understood. However, mutations appear to be relatively specific to aCML, as they are otherwise reported only in few cases of CMML and of systemic mastocytosis with associated eosinophilia [In 2015, recurrent mutations in nophilia .CSF3R gene, initially described in both CNL and aCML [CSF3R mutations are less frequent in aCML, being found in < 10% of patients, and are rare in MDS/MPN-U [CSF3R should prompt a careful revision of the diagnosis to rule out the possibility of CNL, as the molecular status would otherwise suggest.By contrast, mutations in the and aCML , appear DS/MPN-U ,8,27. ThSFSR2, especially in the context of monocytosis, may orient the diagnosis towards CMML, in which this gene is mutated in up to 50% of cases.Additionally, mutations in In conclusion, NGS studies could be useful for confirming the clonality of the disease, especially in cases with mild proliferative features and concomitant inflammatory comorbidities that might mimic a reactive leukocytosis process, and may help in the differential diagnosis among different MDS/MPN categories together with morphologic and clinic data.Moreover, the molecular findings may have crucial relevance in directing personalized therapies, since aCML-associated genetic alterations might be susceptible to specific therapeutic approaches, directly targeting the mutant proteins or their associated pathways .NRAS, JAK2, and CSF3R mutations directly impact on the MAPK and JAK/STAT pathways; SETBP1 encodes a protein named SET binding protein 1 (SEB) that impacts on AKT and MAPK pathways; mutations in the PTPN11 gene result in constitutively activated RAS signaling [Indeed in the last decade, the identification of oncogenic mutations in signal transduction proteins has clarified that specific pathways regulating the proliferation of myeloid lineage cells may be deregulated in leukemia . Severalignaling . InhibitASXL1, RUNX1, EZH2, and TP53 [In addition, in myeloid disorders, and particularly in MDS, mutations in several genes are adverse prognostic factors, namely alterations of and TP53 ,32,33. IFinally, the identification of molecular markers may be useful to monitor minimal residual disease (MRD), as validated in other myeloid diseases and shown in a few studies of aCML ,35.2 IV for 5 days on 28 day cycles) as an off-label prescription. After 3 cycles, the patient achieved a partial hematological response [Due to progressive leukocytosis and monocytosis, a BM assessment was repeated, documenting a stable disease without increase of blasts. However, considering the young age of the patient, the progressive leukocytosis despite cytoreduction and the progressive monocytosis that has been correlated with a higher risk of progression to AML ,23, we oresponse  with a nSETBP1 mutations and the generally poor risk related to SRSF2 alterations in the context of other myeloid neoplasms. Unfortunately, all the mutations detected in this case were not actionable, therefore pegylated interferon alpha (Peg–IFNα) 3 mcg/kg/week was chosen as first line therapy. After 3 months of treatment, a partial response was achieved and maintained over time with no toxicity.The patient was considered unfit for HSCT due to age and to the previous diagnosis of solid neoplasia. Although the patient’s WBC count was only mildly elevated and the Hb and Plts count were well preserved, his long term prognosis was poor due to the general high risk of AML progression related to the diagnosis of aCML itself and the unfavorable prognosis associated to his molecular profile, specifically with JAK2 inhibitors, are currently being investigated. However, up to date, the only curative treatment remains HSCT, an option available for the younger patients only. Participation on clinical trials should be considered in all cases. A possible treatment algorithm is reported in No standard of care is currently available for aCML management. This rare entity of MDS/MPN, as already detailed, is characterized by heterogeneous clinical and genetic features with a very poor prognosis. In addition, due to the low disease incidence and the absence of large randomized clinical trials, the physician’s choices remain challenging. Several treatment strategies validated in other myeloid diseases have been evaluated in aCML, starting from erythropoiesis stimulating agents (ESAs) to improve the anemia and up to cytoreductive drugs to control the proliferation and reduce the blast count , hydroxyurea (HU), or PEG–IFN–α. Given the recent clarification of the mutational landscape of MDS/MPN, different target therapies, including n = 4) or MUD (n = 4) or a syngeneic transplantation from a twin sibling (n = 1) [HSCT is considered as the sole potentially curative strategy for aCML. Data on this approach are scant and most evidence originates from studies including patients with heterogeneous MDS/MPNs. Lim et al. reported a series of 10 patients with MDS/MPN who received HSCT. The 5-year OS and disease-free survival (DFS) rates of the whole cohort were 42.2% and 46%, respectively. Only 2 out of 10 patients were aCML, and, at the median follow-up of 47.5 months, both were alive and in remission . Similar (n = 1) . All pat (n = 1) . A subse (n = 1) . These p (n = 1) . At a meSETBP1 mutation) prognostic factors and to offer HSCT at diagnosis in high risk cases and defer the procedures in the others [No evidence-based information on the best timing of transplantation is currently available, and indications rely on expert opinions. Gotlib et al. proposed HSCT for all candidate patients at the time of diagnosis , wherease others .The choice between these two approaches should also consider the availability of a suitable donor at the time of diagnosis and the presence of a targetable mutation that might be appropriate for an attempt with molecular therapies.In conclusion, allogeneic HSCT may be considered a promising approach for a subset of aCML patients and the new molecular findings may pave the way to a MRD driven approach to treat patients after HSCT; however, a large number of cases are elderly and may be not candidates for the HSCT.BCR/ABL1 positive and negative CML. Out of seven aCML patients, four obtained a clinically meaningful response but their median survival was only 13 months and the 2 year survival rate was 14% [Based on the use of the HMA azacytidine or decitabine in CMML, different studies have tried to use these drugs also in aCML . Kantarj was 14% . More re was 14% ,46,47,48HMA cannot be considered as a standard of care for aCML and their use is off-label. However, these drugs represent an alternative to standard chemotherapy for the pre-transplant cytoreduction in case of a low blast count, as in our clinical case 1, or in the presence of comorbidities. Moreover, given the evidence that the use of HMA as a bridge to HSCT in MDS does not worsen the transplant outcome and OS after transplant, it is tempting to extrapolate this hypothesis also to aCML ,50,51. HHMA may also be considered for patients who are ineligible to transplant that do not carry actionable somatic mutations and are intolerant to HU as an alternative to IFN.The role of AML-like intensive chemotherapy has not been explored extensively, but is usually offered to selected patients with high-risk proliferative clinical behavior as a bridge to HSCT .HU is commonly used to control splenomegaly and leukocytosis. Older studies reported that HU and IFN-alfa are able to induce a complete hematological response, however with short duration ,11. PEG–Several promising targeted therapies are currently being investigated, including the JAK inhibitor ruxolitinib, the SRC kinase inhibitor dasatinib, and the MEK inhibitor trametinib , with manageable or reversible toxicities [NRAS mutation who obtained a near complete hematologic response within several months of treatment with trametinib [CSF3R mutant aCML [Another option for ted mice . These pxicities . A singlametinib . Moreoveant aCML .Finally, there might be a future role for activators of PPA2, a major protein phosphatase which acts as a tumor suppressor, in the setting of aCML with SETBP1 overexpression. In fact, overexpression of SETBP1 protects SET from protease cleavage, increasing the formation of a SETBP1-SET-PP2A complexes that result in PP2A inhibition, promoting proliferation of the leukemic cells. Pharmacological activation of PP2A seems to offer a future therapeutic alternative, since in vitro PP2A restoration by PADs  reverseClinical trials including aCML patients are needed to come to clear treatment recommendation, even if the rarity of disease is an obstacle to the design of studies with high numerosity.At the moment of manuscript preparation, only 2 trials are actively recruiting aCML patients. Both are focused on the blastic phase of aCML. One is a phase I/II trial on a combination of azacitidine, venetoclax, and pevonedistat in patients with newly diagnosed AML secondary to other myeloid diseases including aCML (NCT03862157). The second study is a phase II trial testing the combination of topotecan hydrochloride and carboplatin with or without veliparib in AML secondary to myeloproliferative disorders or aCML in advanced phase (NCT03289910).The diagnosis of aCML is always a difficult task to accomplish. Until now, the diagnosis has been based on morphological grounds only, possibly underestimating the true frequency of the disease. Only recently, new insights in the molecular biology of MDS/MPN syndromes have deepened our knowledge in aCML. However, all the mutations described in aCML so far are neither specific nor diagnostic, being present in a wide range of other hematological malignancies and also in the normal elderly population, even though at a very low VAF. With a more comprehensive molecular profiling, the diagnostic criteria will be hopefully refined in the future to include also genetic features resulting in decreased reliance on absolute cutoffs in blood counts that may vary along with the disease history of the patients. Nonetheless, as discussed earlier, in some cases these alterations can be more than a simple diagnostic test and may allow personalized treatment. Large collaborative efforts are needed to design studies that will provide data so that the management of these diseases will be evidence-driven.


IS and intra-breed structure show that there is some heterogeneity and structure within the breed. However, inter-breed structure results underline that this breed is differentiated from other Creole breeds, because crossbreeding with other breeds was not detected; therefore, we must take advantage of this valuable genetic resource, and ensure its conservation and selection.An individual from a population presents a series of characteristics that differ from the rest and that increase as the kinship relationships are lower; this leads to the fact that two populations that stop exchanging genetic material through mating eventually come to present characters common to all members of each, but different between the two. This was what happened with the animal populations brought to America more than 500 years ago from the Iberian Peninsula in the colonization period, resulting in Creole populations that inhabit the harshest environments of our immense geography. The Creole goat “Chusca Lojana” has adapted to live in the dry forest region of Southern Ecuador, where environmental conditions are warm-dry, with sparse vegetation and a rather irregular topography. In the present study, the intra-breed genetic diversity of this goat is analyzed as well as its genetic relationships with other breeds. Significant FIS value of 0.11. Between-breed genetic diversity among 43 goat populations  showed the following values: FIS = 0.087, FIT = 0.176, and FST = 0.098. Regarding the analysis of the population structure, the results showed that the Creole Chusca Lojana goat population is homogeneous and no genetic separation was observed between the different biotypes (FST = 0.0073). In conclusion, the Chusca Lojana goat has a high genetic diversity, without exhibiting a genetic substructure. Therefore, it should be considered as a distinct population because crossbreeding with other breeds was not detected.The largest population of goats (62%) in Ecuador is in the dry forest region in the south of the country. A Creole goat, named “Chusca Lojana”, has adapted to the dry forest region where environmental conditions are warm-dry, with sparse vegetation. Knowledge of the genetic information of the Creole goat is important to determine intra-racial diversity, the degree of genetic distance among other breeds of goats, and the possible substructure of the population, which is valuable for the conservation of such a species’ genetic resources. A total of 145 samples of the Creole goat was taken from the four biotypes previously identified. Genetic analyses were performed using 38 microsatellites recommended for studies of goat genetic diversity (FAO-ISAG). The results of within-breed genetic diversity showed a mean number of alleles per locus (MNA) of 8, an effective number of alleles (Ae) of 4.3, an expected heterozygosity (He) of 0.71, an observed heterozygosity (Ho) of 0.63, polymorphic information content (PIC) of 0.67, and an F Biodiversity and abiotic factors are responsible for maintaining the balance and stability of ecosystems. According to , biodiveIn the last two decades, however, such genetic heritage has been jeopardized and more than one million species have become endangered ,3. RegarGoat farming is of great significance for the economy of rural populations worldwide, especially in developing countries due to the rusticity of goats ,8. GoatsHair samples were obtained from 145 Chusca Lojana goats of four different biotypes, the same ones that differ from each other in the size and shape of the ears  and in the absence of horns (“muco” biotype). Sampled individuals belonged to different herds and locations and kinship was minimized, so genetic variability of sampling was ensured. Samples were collected in paper envelopes with individual information and stored at room temperature until genetic analyses were performed at the Laboratory of Applied Molecular Genetics of the University of Cordoba, Spain. Sampling was carried out by qualified veterinarians during routine technical assistance, so the approval of an animal ethics and welfare committee for biological sampling collection was not required. The genomic DNA of the hair follicle samples was extracted according to the methodology described by .BM1258, BM1329, BM1818, BM6506, BM6526, BM8125, CSRD247, CSRM60, CSSM66, ETH010, ETH225, HAUT27, HSC, ILSTS008, ILSTS011, ILSTS019, ILSTS030, ILSTS087, INRA005, INRA006, INRA023, INRA063, INRA172, MAF065, MAF209, McM527, MM12, OarFCB011, OarFCB020, OarFCB048, OarFCB304, SPS115, SRCRSP05, SRCRSP08, SRCRSP23, SRCRSP24, TGLA053, and TGLA122. After amplification by the polymerase chain reaction (PCR) technique, size separation of the amplified fragments was carried out by polyacrylamide gel electrophoresis in an ABI 3130XL automatic sequencer by means of Genescan® 400HD ROX Size Standard . Fragment analysis and allelic typing were performed using Genescan Analysis® 3.1.2  and Genotyper® 2.5.2 software , respectively  for genetic diversity studies in goat species. The microsatellites included were ectively .IS fixation index values with a 95% confidence interval were calculated with the GENETIX v software 4.05 [The MICROSATELLITE TOOLKIT software for Excel  was usedare 4.05 . The Harare 4.05 , which aare 4.05  and Bonfhttps://biogoat.jimdo.com/razas-breeds/).To obtain the genetic differentiation, structure, and distance information data of the Chusca Lojana goat was compared with data of other 42 goat populations worldwide, which included some native Creole breeds from Spain, Europe, America, and Africa . Data onIT, FST, and FIS [Wright’s F statistics, namely F and FIS , were ca and FIS . A Facto and FIS  was dete and FIS . With th and FIS  to graphSA) were calculated [The genetic distances between individuals . After Bonferroni correction, only the INRA023 and SRCRSP24 markers were unstable compared with the Hardy–Weinberg equilibrium of the population. Only the INRA005 and SPS115 markers showed a homozygous defect, but their FIS values were not significant. Half of the microsatellites (n = 19) showed a significant excess of homozygosity, and the other microsatellites (n = 19) displayed FIS values that were not significantly different from 0.Shown in IS with a 95% confidence interval and 1000 randomizations was significant (FIS = 0.1099 (0.08773–0.12549)), which indicates that the population could deviate from the Hardy–Weinberg equilibrium.The mean number of alleles (MNA) is an indicator of the genetic variability within populations. The MNA is 8.26 in the Chusca Lojana goat, and with an average Ae value of 4.25. We assessed the genetic diversity within a population by calculating the proportion of heterozygous individuals or heterozygosity. In ST = 0.0073).To make a preliminary assessment of the population’s homogeneity, a tree diagram of the genetic distances between individuals was designed , in whicA more complex analysis of the population structure was also carried out using STRUCTURE v. 2.1. This software  allowed IS = 0.0871 (0.065–0.116), FIT = 0.176 (0.154–0.205), and FST = 0.0978 (0.089–0.108).The genetic differentiation among the 43 populations of goats included in the present study was high, with the following statistical values for F: FThe results of the Correspondence Factor Analysis  show thaST values between pairs of goat populations are shown in The Reynolds’ genetic distance data and the FIn the graphic representation of Reynolds’ genetic distances in a network dendrogram , it can Shown graphically in The characterization process is the first step of the zoogenetic resources conservation programs. So, studies of this nature are generally recommended to save locally adapted breeds from extinction .The high genetic diversity of the Chusca Lojana breed is demonstrated by the mean number of alleles (MNA = 8.3) and the effective number of alleles (Ae = 4.3). These values are similar to those obtained for the Spanish goat of the USA  and higher than those of the Creole goats of Argentina, Bolivia, Brazil, Colombia, Cuba, Peru, and Venezuela . It is noteworthy that the Galapagos Creole goat displayed lower MNA and Ae values , and the Creole Goat of Paraguay showed higher MNA and Ae values  [IS value of the Chusca Lojana breed was significant (0.1098) and higher than the mean of the corresponding values reported for the other American goat populations [IS value could be due to non-random mating within the breed. Significant inbreeding levels and deviations from the Hardy–Weinberg equilibrium can be shown in a short time if no measures are taken.The Chusca Lojana breed exhibited higher expected (He = 0.71) and observed heterozygosity values (Ho = 0.63) than the mean of the corresponding values for the other American Creole goat populations . The only exception was the Paraguayan Creole, which showed higher He and Ho values . The genulations ,27, beinIS of the 57 Asian breeds was lower (0.073) and the FIT = 0.191 was similar to the corresponding values in the present study.Interestingly, the Chusca Lojana breed showed higher genetic diversity  than reported for 57 native goat breeds in Asia  . HoweverST) of the Chusca Lojana breed and the other 42 Creole and transboundary breed populations was lower (FST = 0.098) than the values reported for 24 Creole and three cross-border breeds in America , for the Creole breeds of Asia (FST = 0.127) [ST = 0.112) [ST of the Chusca Lojana breed was higher than the values obtained for goats from Northern Europe and Central-Eastern Mediterranean (0.07) [The genetic differentiation  , and for= 0.112) . On the n (0.07) , West Asn (0.07) , and Afrn (0.07) .By graphic representation of Reynolds’ genetic distances, it could be demonstrated that results in the present study are in accordance with the data previously reported , in whicFinally, both the work carried out by  and the The Chusca Lojana goat breed exhibits a high genetic diversity, with a heterogeneous structure inside the population; but, the existence of four different biotypes empirically admitted inside the breed was not supported by our genetic results. The Chusca Lojana breed belongs to the genetic group of the American Creoles, descendants of the of the Iberian populations imported by the Spanish colonizers five centuries ago. No genetic introgressions from the most important international breeds  were detected in the Chusca Lojana breed.The Chusca Lojana breed is a valuable zoogenetic resource, well adapted to its difficult environment; its conservation and selection should be encouraged, and actions should be taken to avoid future genetic erosion.


Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations , comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.The use of controlled mixed inocula of  Saccharomyces cerevisiae, which quickly dominates the fermentation of grape must in wine production. However, in the last years, there has been an increasing interest in studying other fermentative yeasts, which are generally referred to as non-Saccharomyces yeasts, due to the ability of some of them to improve the complexity of wines by increasing the concentration of aromatic molecules, such as terpenoids, esters, higher alcohols or other molecules of interest, such as glycerol [Saccharomyces yeasts are known to spoil wines and cause stuck fermentations [Saccharomyces yeasts are used as starters together with S. cerevisiae, they are able to participate in the fermentations until late stages, and produce wines with the previously mentioned characteristics [Metschnikowia pulcherrima, Torulaspora delbrueckii and Lachancea thermotolerans. All of them showed the ability to reduce ethanol content, mainly M. pulcherrima [M. pulcherrima has been described as a yeast that makes wines fruitier and fresher, positively modulating the wine aroma profile [L. thermotolerans increases wine natural acidity due to increased lactic acid production [T. delbrueckii has a positive influence on the overall impression of the obtained wines, improving the aroma quality and the varietal character [Alcoholic fermentation of grape must is mainly driven by glycerol ,3,4,5,6.glycerol ,8,9. In ntations ,11. Howeeristics . Among tcherrima ,15,16,17 profile ,18. L. toduction ,19,20 anharacter ,22.S. cerevisiae as starters produces wines with improved qualities, their interactions at the microbiological level are still not completely understood. Very early studies of alcoholic fermentation confirmed that S. cerevisiae quickly dominates the fermentation process, resulting in the disappearance of the non-Saccharomyces yeasts, which was initially attributed to different ethanol and SO2 sensitivities of the latter [Saccharomyces yeasts to survive until the end of the fermentation, with a broad array of interactions among different yeasts [S. cerevisiae is responsible for the premature death of non-Saccharomyces yeasts [Saccharomyces and S. cerevisiae yeasts can also cause different responses in the cells. In fact, several studies have shown early growth arrest of different non-Saccharomyces yeasts in co-fermentations with S. cerevisiae due to a cell–cell contact mechanism [Therefore, although it has been proved that the use of some of these yeasts together with e latter . Howevert yeasts ,12. Indet yeasts ,25. Froms yeasts ,27,28. Fechanism ,30,31,32Saccharomyces yeasts, but not all can be differentiated with this method, as not all the species present different colony morphologies [The detection and quantification of the yeasts involved in wine production have been studied for many years and with different methodologies. The most widely used method is based on the culturability on different solid media. The use of differential media, such as Wallerstein Laboratory Nutrient (WLN) medium, allows the differentiation of some non-hologies . Indeed,hologies ,35,36,37hologies ,39. In ahologies ,41, but hologies ,43.As mentioned before, interactions among yeasts during alcoholic fermentation can lead to cell death or growth arrest. If this occurs, the qPCR results will not be accurate enough since the test quantifies the total DNA, not differentiating between dead and living yeasts. To solve this problem, some studies have applied reverse-transcription PCR (RT-PCR), a technique that amplifies the genetic material only from viable cells ,44,45. H2 in order to eliminate competitive advantages for S. cerevisiae, and to determine the VBNC population. For that reason, we simultaneously inoculated a synthetic must with S. cerevisiae and three non-Saccharomyces yeasts commonly used in mixed wine industrial fermentations , and analyzed yeast dynamics and viability for each species by PMAxx-qPCR and compared the results with those from non-dyed qPCR and colony counting.Our aim in this study was to analyze the population dynamics of mixed alcoholic fermentation without the use of SOS. cerevisiae QA23  (Sc), T. delbrueckii Biodiva  (Td), L. thermotolerans 1  (Lt) and M. pulcherrima CECT 13131 (Mp) isolated from grape must from the Priorat region (URV collection) [w/v) glucose, 2% (w/v) yeast extract, 1% (w/v) peptone and 1.7% (w/v) agar) and on Wallerstein Laboratory Nutrient (WLN) agar  from frozen stocks, and one colony was used to develop the inoculum. Before starting fermentations, routine species confirmation of the different strains was performed by restriction analysis (PCR-RFLP) of 5.8 S-ITS rDNA [Four yeast species were used in this study: lection) . StrainsITS rDNA .5 cells/mL of each strain were simultaneously inoculated in 230 mL of a synthetic must [6 cells/mL. Fermentations were performed in triplicate and incubation was at 22 °C with stirring (120 rpm).Single colonies of each strain were grown individually in YPD liquid medium for 24 h at 28 °C. Cells were counted in a Neubauer chamber and 5 × 10tic must , containFermentations were monitored by measuring must density  and optical density at 600 nm . Fermentations were considered to be finished when residual sugars were determined to be less than 5 g/L .The population dynamics were monitored by plating samples at different points of fermentation on two solid media. Briefly, samples were serially diluted in sterile water and the number of colony-forming units per milliliter (CFU/mL) was determined by plating 100 μL of the corresponding dilutions on YPD for total yeast population and on WLN for the morphological differentiation and counting of the four species present in the sample.TM viability dye  was used. First, we validated the effectiveness of this dye using living cells and heat-treated cells (as dead cells).To better differentiate between living and dead yeast cells and to ensure that the qPCR only amplifies living cells, the PMAxx6 cells/mL) were centrifuged at 6000 rpm for 5 min and the pellet was resuspended in 1 mL of sterile distilled water. One of the aliquots was subjected to heat shock (95 °C for 10 min) and both were stained with PMAxx according to the manufacturer’s protocol, with the modification of the cross-link time. Briefly, the PMAxx dye (25 µM) was added to the sample, followed by an incubation time of 10 min in the dark. Then, based on previous studies [One yeast colony was inoculated in YPD liquid medium and incubated overnight at 28 °C. Then, two aliquots of 1 mL sample . Then, DNA from both aliquots was extracted as described below.Saccharomyces species were published by García et al. [S. cerevisiae primers were published by Hierro et al. [TM Premix Ex TaqTM II  according to the manufacturer’s instructions in a QuantStudioTM 5 real-time PCR instrument . The amplification process included an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturing at 95 °C for 5 s and annealing at 60 °C for 30 s. Cycle threshold (Ct) was determined using the Standard Curve application . Primer specificity was checked by performing the same qPCR with DNA of all species involved in the study. Milli-Q water  was used as a negative control of amplification (NTC).DNA was extracted using the DNeasy Plant Mini Kit according to the manufacturer’s instructions . Primers used to amplify non-a et al. , S. cereo et al.  and the o et al.   were mixed with DNA extracted from 106 cells/mL of dead cells from the same species. Additionally, in living/living mixed cultures, the same serial dilutions of DNA from living cells were mixed with DNA extracted from 107 cells/mL of living cells from another yeast species . According to the Clinical and Laboratory Standards Institute (CLSI), the LoD is the lowest amount of analyte in a sample that can be detected with probability, although perhaps not quantified as an exact value (Ct < 40). The LoQ is the lowest amount of measurand in a sample that can be quantitatively determined with acceptable precision and stated, acceptable accuracy, under stated experimental conditions (Ct < 30).The limit of detection (LoD) and quantification (LoQ) by qPCR of living cells treated with PMAxx were calculated in pure and mixed cultures. For the latter, two different types of combinations were prepared for each yeast species. In living/dead mixed cultures, tenfold serial dilutions of living cell DNA . Results are expressed as the mean ± SD. Student’s t-tests were applied to analyze differences between living and dead cells and the influence of PMAxx treatment (p < 0.05).All analyses were performed using GraphPad PrismPMAxx is an optimized version of PMA, a fluorescent photoaffinity dye that binds covalently to DNA after photoactivation, inhibiting its amplification by PCR. As PMAxx can only enter cells with compromised or damaged membranes, but not intact cells, predominantly, DNA from living cells will be detected by qPCR. In our study, dead cells treated with PMAxx showed the maximum possible Ct reduction , while for Lt and Mp, no differences in LoQ were observed between PMAxx-treated and untreated cells. The LoD was one log lower in all cases  to test if PMAxx-qPCR could be used to monitor the viable population dynamics of each species during mixed fermentation. The results of PMAxx-qPCR were compared with those from non-dyed qPCR and with a culture-dependent technique, using YPD and WLN differential media.7 CFU/mL), but since day 2, each species followed different dynamics. Td was the species with the highest number of CFU/mL counted on WLN plates, in all sampling points, with its maximum growth at day 3 (6.17 × 107 CFU/mL). Sc colonies were detected throughout fermentation, but, surprisingly, Sc was not the main strain at the end of fermentation. In contrast, Mp and Lt colonies were not detected at all sampling points. Hence, Mp suffered a drastic fall in plate counts starting at day 3, so that the last colonies were observed at day 4. Lt was the species with the fastest decrease in plate viability, as no colony was observed after day 3; however, surprisingly, at day 7, some colonies were recovered in two out of three replicates. The absence of Lt colonies at days 4 and 6 showed that colony counting is not a suitable method for the detection of minority strains. The sample dilution necessary to have a proper number of colonies in a plate (30–300 CFU/mL) rules out the possibility of detecting low-abundance strains.Mixed fermentations were completed in 7 days a. First,Plating results were then compared with qPCR analysis. For qPCR, samples of days 0 (1 h after inoculation), 1, 3, 4, 6 and 7, treated and untreated with PMAxx, were analyzed . In a geS. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima) currently used in the wine industry, in comparison to non-dyed qPCR and classical microbiological methods.The study of microbial population in alcoholic fermentation has been traditionally performed by the classical microbiological colony-counting technique ,23. HoweSaccharomyces species used in this study are all commercially available as active dry yeast starters and are used in mixed or sequential fermentations with S. cerevisiae due to the positive characteristics given to the final wine [The non-nal wine ,18,19,62When analyzing the population dynamics of spontaneous or mixed fermentations, the classical solid medium used to study the cell population presents important drawbacks to properly distinguish and count all the species present ,36, and Opposite to colony counting, the use of qPCR allowed the detection of the four yeast species throughout the fermentation. In agreement with previous studies, the cell population numbers obtained by an independent-culture technique, such as qPCR, were higher than the results obtained by plating ,39,61,636). In this study, the interference with DNA from different living cells (107) was also tested, and again, linearity of standard curves was not influenced. In addition, LoD and LoQ were calculated in the presence of both exogenous DNA molecules and these limits were not changed in any condition.To achieve an exact quantification of viable cells, samples were treated with PMAxx, so that only DNA from cells with an intact membrane, impermeable to this dye, can be amplified. Several studies have proven that this kind of dye, known as a viability dye, is useful to assume that the amplified population can be considered as viable cells ,49,53,55Brettanomyces bruxellensis, obtaining better results by PMA-qPCR than by colony counting, qPCR or even RT-qPCR.The application of PMAxx-qPCR allowed us to obtain a more accurate determination of the population dynamics. At all sampling points analyzed, except for time 0, the quantification obtained by PMAxx-qPCR was closer to the results of colony counting, while non-dyed qPCR overestimated the population in all cases. As we have previously noted, this may be due to the ability of PMAxx to bind the DNA from dead cells, impairing its amplification, so that qPCR will mainly amplify and detect the DNA from viable cells ,45,50. TS. cerevisiae changes due to cultivation in a hyperosmotic medium (high NaCl concentration); this change allows the entry of several DNA-binding dyes into cells. Grape must is a hostile and stressful medium for yeast cells [GPD1, an osmotic stress gene, is activated within the first hour after inoculating grape juice from a stationary preculture [Nevertheless, some discordant points were detected. One of them was the low number of viable cells obtained by PMAxx-qPCR after one hour of must inoculation compared with colony counting or non-dyed qPCR. Capusoni et al.  showed hst cells ,66,67. Weculture . In thisS. cerevisiae and L. thermotolerans in a VBNC state during fermentation. This behavior has been widely described by several authors in different bacteria and is considered as a survival strategy that permits resilience to unfavorable environmental conditions [Saccharomyces yeasts. Thermosonication in B. bruxellensis [Cryptococcus neoformans [S. cerevisiae supernatant produced the loss of culturability of Hanseniaspora uvarum, M. pulcherrima and T. delbrueckii, suggesting that the presence of some antimicrobial metabolites could induce the VBNC state. More specifically, Branco et al. [S. cerevisiae caused the loss of culturability in H. uvarum. Regarding S. cerevisiae, for the first time, our study evidenced the presence of the VBNC state in mixed fermentation. This VBNC population was found in a high proportion (more than 85%) from the middle to the end of fermentation, reaching 93.74 ± 10.07%. Previous studies have demonstrated that S. cerevisiae is able to enter into a VBNC state under SO2 and heat shock stress [S. cerevisiae and L. thermotolerans modifies yeast metabolism and the exometabolome, and cell contact can also influence S. cerevisiae viability. In fact, common winemaking procedures include the use of SO2 to prevent the growth of unwanted microorganisms, among them, non-Saccharomyces yeasts. Industrial wine S. cerevisiae yeasts are known to present resistance to SO2, and this is one of the mechanisms of inducing VBNC status in non-Saccharomyces yeasts [S. cerevisiae during the winemaking process. For this reason, in the present work, SO2 was not used to prevent the bias that would be introduced by its addition. Furthermore, the use of SO2 in the wine industry has been challenged due to side effects in human health; thus, our conditions are also useful in this new SO2-free scenario.However, the most important aspect revealed by the use of this viable dye was the existence of nditions ,69,70. Tnditions . Some stnditions ,72 and eellensis  and hypooformans  have trioformans  showed to et al.  showed hk stress ,76. In ak stress  demonstrs yeasts  and induS. cerevisiae was revealed for the first time during mixed alcoholic fermentation. Further research is still required to understand how the interactions between S. cerevisiae and non-Saccharomyces yeasts impact their physiological and metabolic status and which conditions produced during fermentation are causing this state.In summary, the use of the viable dye PMAxx coupled with qPCR is a reliable, specific and fast method for monitoring population dynamics in mixed fermentations. In addition to detecting minority yeasts until the end of fermentation, the presence of a VBNC state in


Lung cancer remains the most common cancer in the world. The genetic polymorphisms  were shown to be strongly associated with the risk of lung cancer. Our study's aim is to elucidate whether these polymorphisms predispose Eastern Algerian population to non-small-cell lung cancer (NSCLC). To date, no study has considered this association in the Algerian population. This study included 211 healthy individuals and 144 NSCLC cases. Genotyping was performed using TaqMan probes and Sanger sequencing, and the data were analyzed using multivariate logistic regression adjusted for covariates. The minor allele frequencies (MAFs) of TERT rs2853669, CHRNA5 rs16969968, and OGG1 rs1052133 polymorphisms in controls were C: 20%, A: 31%, and G: 29%, respectively. Of the three polymorphisms, none shows a significant association, but stratified analysis rs16969968 showed that persons carrying the AA genotype are significantly associated with adenocarcinoma risk . Smokers with an AA allele have a larger risk of lung cancer than smokers with GG or GA genotype , which is not the case of nonsmokers. Our study suggests that CHRNA5 rs16969968 polymorphism is associated with a significant increase of lung adenocarcinoma risk and with a nicotinic addiction. Lung cancer (LC) remains the most common cancer in the world, both in terms of new cases  and deaths each year (1.7 million deaths 18.4%) , 4. In aTERT, encoded by the TERT gene, is one of the major components of telomerase; it is a reverse transcriptase that can add a nucleotide sequence (5′-TTAGGG) to telomere end . The proEscherichia coli mutant strain deficient in 8-oxoG [The OGG1 gene is located at chromosome 3p26.2. Its protein is a DNA repair enzyme, having both DNA glycosylase and AP lyse (apurinic or apyrimidinic site lyase) activities to remove 8-hydroxyguanine (8-oxoG) lesion produced by reactive oxygen species . The rs1n 8-oxoG . It has n 8-oxoG –18.CHRNA genes code for proteins that form receptors which bind to nicotine and its metabolites . In a laIn our study, we were interested in SNPs of three different genes, which were selected based upon GWAS and meta-analyses of multiple populations TERT , OGG1 (rs1052133), and CHRNA5 (rs16969968). The aim of our study is to investigate how these SNPs can modify the risk of LC in the Eastern Algerian population. It is important to mention that our population, to the best of our knowledge, has not been the object of any molecular study concerning the LC till now, and thus this study, as any other pioneering work, had to deal with a relative scarcity of data.Although this study has been conducted within various population centers in Eastern Algeria which thus encompass several ethnic groups, Arabs and Central Algeria Berbers, yet the population mixing which took place at an accelerated pace after Algeria's independence, the standardization of lifestyles, the rural exodus, all concurred to homogenize the genetic stock to a large extent . FurtherWe have performed in this work a case control study of 144 LC patients and 211 healthy controls. The patients were first diagnosed with NSCLC between June 2015 and July 2016. They came from a wide zone spanning five Wilayas  in Eastern Algeria between governmental University Hospitals  and private clinics. There was no restriction on gender, histologic subtype, or stage with the exception of patients who refused to participate in the study. Diagnosis was made by cytological imaging and histopathological examinations. We feel confident as explained previously that our samples are largely homogeneous. In addition to that, the democratization of access to medical treatment, freely provided to all, as shown in the fact that city dwellers and those from rural areas are equally represented in the various patient samples, allowed us to make the claim of genetic homogeneity of the samples with a good degree of confidence.The control group comprised individuals with no history of cancer and without lung-related disease to avoid possible interference from overlapping genes. They were chosen from a pool of volunteers in the blood transfusion centers during the same period of patients without restriction of age or gender. All subjects were Algerians residing in Eastern Algeria.We used a standard epidemiological questionnaire submitted to patients and controls, and we used the medical files to collect personal data, including residential region, age, gender, profession, smoking status, symptoms, family history of cancer, histologic subtype, and stage TNM.Our research has been approved by the local Ethics Committee. The use of human blood sample and the protocol in this study strictly conformed to the principles expressed in the Declaration of Helsinki, and informed consent was obtained from all participants or from their family members.Genomic DNA was extracted from six to eight milliliters of peripheral venous bloods, which was collected into a tube containing ethylene-diamine-tetraacetic acid (EDTA) from each participant. We used standard protocols following the salt extraction procedure. The DNA concentration was measured with NanoDrop One spectrophotometer. For genotyping rs16969968 and rs1052133, we used Polymerase Chain Reaction (PCR) TaqMan; with ViiA 7 (Applied Biosystems) and TaqMan® SNP Genotyping assay kits (Applied Biosystems), we used the standard protocol recommended by the manufacturer.The promoter TERT was amplified by PCR, and the PCR primers used were as follows: 5′ACCGTTCAGTTA GCGATTTCCCACGTGCGCAGAGGAC3′ (forward), and R: 5′CGGATAGCAAGCTCGTCTCCCAGTGGA TTCGCGGGC3′ (reverse). Then, it was genotyped with Sanger sequencing in both the forward and reverse directions using the Big Dye Terminator Chemistry V.I and an automated fluorescent sequencer, an ABI 48 Capillary Array Sequencer (Applied Biosystems 3730). Data management and analysis were done using SeqScape. For TERT sequencing, we could do sequencing for only 126 cases and 94 controls.http://www.ncbi.nlm.nih.gov/SNP/) and a study power fixed at 80%. The Hardy–Weinberg equilibrium of the genotype distributions is evaluated in the control subjects using a function from R package “Hardy–Weinberg” (Version 1.6.1).To estimate sample size, we used functions from the R package “epiCalc” (Version 2.9.0.1) with a minor allele frequency data obtained from NCBI  with the corresponding confidence intervals (95% CI). The ORs are also performed for a heterozygote model , homozygote model (Aa vs. AA), recessive model (AA+Aa vs. aa), and dominant model (AA vs. Aa+aa). We used Pearson's chi-square test to compare distributions of demographic variables, smoking status, and genotypes of the three genes between cases and controls. To calculate ORs and 95% CI, unconditional logistic regression analyses are performed. In addition, multiple unconditional logistic regression analyses with adjustment for possible confounders  are performed to calculate adjusted ORs and 95% CI. Statistical tests along with ORs and corresponding CIs are done using the publicly available packages of R Project for Statistical Computing (R version 3.5.1) and especially functions from packages MASS and questionr (Version 0.7.0). All statistical analyses are two sided, and the significance level is set at p < 0.01). Thus, the cases and controls were incompletely matched in this study population. Among the cases, 66% were diagnosed with adenocarcinoma and 34% with squamous cell carcinoma. Approximately 73% of case subjects and 37% of control subjects were ever smokers. As expected, case subjects had a significantly higher level (p = <0.01) of smoking than control subjects.To test the association between TERT, OGG1, and CHRNA5 gene polymorphisms and the risk of LC, we conducted a case-control study consisting of 144 NSCLC cases and 211 controls enrolled in four University Hospitals of Eastern Algeria . The distributions of age, gender, smoking history, and histology type among the study subjects were summarized in p values are as follows: 0.15 for TERT, 0.49 for OGG1, and 0.15 for CHRNA5.The distribution of TERT, OGG1, and CHRNA5 gene polymorphisms in cases and controls are presented in After sequencing the promoter of TERT gene, the two SNPs could not be found in this population , but we observed the presence of the rs2853669 polymorphism with a high frequency.p = 0.02 and p = 0.03, respectively). After adjustment for the sex and age, the signification becomes marginal (data not shown), and after adjustment for the sex, age, and tobacco consumption factors, we found that the correlation vanished , and pAdj = 0.25, OR = 1.75, CI = (0.68–4.69), respectively).The minor allele frequencies (MAFs) of TERT rs2853669, OGG1 rs1052133, and CHRNA5 rs16969968 and polymorphisms in cases/controls were C: 24%/20%, G: 26.4%/29%, and A: 35.7%/31%, respectively. The genotype distribution of TERT and OGG1 was not significantly different between LC and control groups in all models, although the genotype distribution of CHRNA5 was significantly different in the recessive and homozygote modes . Among males, the homozygote genotype CC increase the risk of LC than TT or TC genotypes with p = 0.03 . After adjustment for age, sex, and smoking status, the signification become marginally significant ).The OGG1 rs1052133 did not show any signification with stratified factors, except for age (>60 years), which reveals an association with the increase of LC for the persons having a minor allele (GG), in comparison with person with CC genotype ; after adjustment for age and sex, the signification persists with pAdj = 0.03, ORAdj = 3.91, and CI = (1.24 − 17.34). For the stratified analysis according to the histological type, we observed that the persons with AA allele have preferentially an adenocarcinoma rather than squamous type under the recessive and homozygote models p = 0.003 and p = 0.005, respectively. This risk was not affected by adjusting for other factors  pAdj = 0.03, ORAdj = 2.55, and CI = (1.07 − 6.35).As for CHRNA5 rs16969968, we observed an increasing risk for LC among males with a recessive genotype (AA) compared with those having GG or GA genotype with As for the stratified analysis by sex, smoking, histological type, and age, according to alleles, no significant association was found.High incidence and poor prognosis of LC make it a major health problem worldwide. Although the LC is linked to environmental exposure to carcinogens, especially cigarette smoking, only some smokers develop LC, which suggests that there is an interindividual difference in susceptibility to the disease. In this study, polymorphisms in three genes involved in the metabolism of carcinogens or in the repair of damaged DNA in lung cells, TERT (rs2853669), OGG1 (rs1052133), and CHRNA5 (rs16969968), were examined for association with NSCLC risk in a case-control study of 144 patients and 211 control subjects of Eastern Algerian population in an attempt to explain this interindividual difference.In our population, none of these three genes shows an influence on the LC risk, except the CHRNA5 rs16969968 SNP, which was significantly different between cases and controls, but after adjustment, the signification disappears. These results are in accordance with the findings of numerous studies but contradictory to others , 29. TheFor TERT gene, after sequencing its promoter, we identified in our population the rs2853669 polymorphism. The rs2853669 MAFs in our control group is C = 20%. According to literature and HapMap data, there is no big difference between the MAFs of rs2853669 in Caucasian and Asian populations, C: 26%-37% , 31. Ovep = 0.46). Two previous studies found that this SNP could have a protective effect for LC [For the OGG1 rs1052133, previous studies have shown that homozygous carriers of the variant appear to have reduced repair capacity toward oxidized DNA lesions, and previous evidence indicated higher levels of 8-oxoG in lung tissue of LC patients than in lung tissue of patients without cancer , 35. Numt for LC , 38. Ovet for LC , 39–41. t for LC . It seemt for LC , 43.In our stratified analysis by histological type, gender, age, and smoking habits, only the age showed a marginally significant elevation of risk of NSCLC for persons more than 60 years old and carrying a recessive genotype GG. However, results obtained by Hung et al. were not in agreement with ours .Our findings could not explain the significantly lower capacity to incise 8-oxoG by leukocytes of NSCLC patients in comparison to healthy controls. A possible explication is that OGG1 gene polymorphisms are only one of many parameters that might affect OGG1 activity. Another explanation as suggested by Lee et al. is that the GG genotype is deficient in the repair of oxidatively generated DNA damage only under conditions of cellular oxidative stress . Howeverp = 0.16). Thus, our data do not support an important role for the SNP in NSCLC risk. However, previous studies on different ethnic groups, predominantly Caucasian, showed a rather important association between rs16969968 and LC risk.The rs16969968 CHRNA5 polymorphism was the subject of a large number of investigations , 26, 45.p = 0.03) under the recessive model. Falvella et al. reported that CHRNA5 mRNA levels are upregulated 30-fold in ADK compared with normal lung tissue in individuals carrying AA genotype compared with those having GG genotype [When stratified according to histologic subtype, rs16969968 was associated with LC risk in ADK but not in SCC , CHRNA5 (rs16969968), and OGG1 (rs1052133) in Eastern Algerian population, and has shown that the rs16969968 CHRNA5 has a significant association with adenocarcinoma LC, and interestingly that the risk is important for smoking patients carrying the AA genotype.These significant and novel results of our study have however some limitations. First, there is the limited sample size used in the study and the mismatch of our population in sex, age, and smoking, which could result in some bias in the results . Secondly, as the sample was not too large, the statistical power to detect the modest effect of three potential functional SNPs in stratified analyses is relatively low. Besides this, there were some missing information on the smoking status for some people and the duration and intensity of the nicotinic addiction tests. In any case, to fully apprehend the difference in the genetic background of the various North African populations will require more studies, and some of the results reported here may help bring new perspectives on the topic.


Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed  The secondary structure of mRNA is important for many translation related processes in bacteria and bacteria-derived eukaryotic organelles. This includes the efficiency of translation initiation ,2,3,4,5,It has been proposed that changes in mRNA secondary structure regulate translation in plastids, i.e., plant organelles derived from cyanobacteria ,18,19,20cis-elements, which are essential for translation initiation, accessible to the ribosome, and thereby activates or upregulates translation.Compared to the intrinsic mRNA features determining the efficiency of translation initiation, we understand much less about the molecular mechanisms regulating translation in plastids. One hypothesis is based on in vitro findings that some RNA-binding proteins can alter the structure of the translation initiation region of their target mRNAs in a way which activates translation initiation ,18,19,20Arabidopsis thaliana plants to high light. In higher plants, this condition is known to induce the translation of the plastid-encoded psbA mRNA (encoding the D1 subunit of photosystem II) on the level of translation initiation ; 16 h dark; 4 h high light [1200 µE m−2 s−1]; and then the leaf material was harvested. The temperature of the growing chamber was set to 20 °C, but owing to the heat emitted by the lamps, the leaves were exposed to temperatures of up to 30 °C. Young leaves with a maximum length of 20 mm were harvested into liquid nitrogen (rosette diameter at this growth stage was 68 ± 3 mm). Plants treated the same way were used for ribosome profiling and RNA-seq  actinic light (81 μE m–2 s–1). In an actinic light phase, saturating light pulses were applied at 20-s intervals. Results were calculated for the last saturating pulse during the actinic light period. Maximum quantum yield of photosystem II (Fv/Fm) and electron transport rate (ETR) parameters were calculated as described previously . In . In psbA, HCF173 , HCF244 , HCF173 , and LPE, HCF173 , whereas, HCF173 . Further73 . Th. ThpsbA  species . HCF244  species ,87. If Hlimation , indicatnslation . This wo3, we demonstrated that the degree of secondary structure of the Shine-Dalgarno sequence and the start codon in the psbA mRNA is reduced in vivo under high light conditions, and that this correlates with increased translational efficiency (psbA translation involves the modulation of mRNA secondary structure by protein binding.Using DMS and a SHAPE reagent, NAI-Nficiency . This coficiency  36]. Th. Th3, werbcL is an example of a gene with a strong SD (rbcL itself is regulated remains unknown. It is possible that distinct mechanisms for regulation of translation initiation exist, as plastids use two distinct mechanisms for start codon recognition [There are indications that such a mechanism is used by other genes: in the case of genes with weak SDs, the change in mRNA secondary structure at the SD correlates with the change in translation efficiency B. Interetrong SD  and heretrong SD . How traognition ,8.psbA (rbcL (psbA, the SD and start codon of rbcL were not paired to a lower extent in high light. Furthermore, the higher accessibility of the SD of psbA in high light was observed both with the DMS-MaPseq and the SHAPE-seq approach (It is important to note that the comparison of the structural changes in the translation initiation regions of psbA  and rbcLbA (rbcL  indicateapproach , althougapproach  comparedapproach .psbA and other plastid genes with weak SDs are in agreement with reports for E. coli that translation efficiency is determined by the extent of RNA secondary structure at the SD [psbA and other genes with weak SDs.The results for t the SD ,5. In bat the SD ,14, bindt the SD ,16, and t the SD . Synthett the SD . Our res


This study was conducted to provide better insights into the demographic and epidemiological characteristics of scorpion envenomation in an endemic area in Algeria and to identify the model that best predicted daily scorpion sting counts.Daily sting data from January 1, 2013 to August 31, 2016 were extracted from questionnaires designed to elicit information on scorpion stings from the two emergency medical service providers in Touggourt, Algeria. Count regression models were applied to the daily sting data.A total of 4,712 scorpion sting cases were documented, of which 70% occurred in people aged between 10 years and 49 years. The male-to-female ratio was 1.3. The upper and lower limbs were the most common locations of scorpion stings (90.4% of cases). Most stings (92.8%) were mild. The percent of people stung inside dwellings was 68.8%. The hourly distribution of stings showed a peak between 10:00 a.m. and 11:00 a.m. The daily number of stings ranged from 0 to 24. The occurrence of stings was highest on Sundays. The incidence of scorpion stings increased sharply in the summer. The mean annual incidence rate was 542 cases per 100,000 inhabitants. The fitted count regression models showed that a negative binomial hurdle model was appropriate for forecasting daily stings in terms of temperature and relative humidity, and the fitted data agreed considerably with the actual data.This study showed that daily scorpion sting data provided meaningful insights; and the negative binomial Hurdle model was preferable for predicting daily scorpion sting counts. Scorpion stings currently constitute a public health concern in many arid, semi-arid, or Saharan regions throughout the world within the stripe of 50° latitude, both south and north. Approximately 2 billion people are estimated to live in areas at risk for scorpion stings. Each year, an estimated 1.2 million people are victims of scorpion stings worldwide. Second only to snake-bites in terms of venomous animal-related human fatalities, scorpions are responsible for an estimated 3,000 deaths each year. However, these estimates are limited to the few countries that have a reliable system for scorpion sting epidemiological surveillance [Androctonus australis stings [Owing to its climatic and geographic conditions and ecological characteristics, Algeria houses a diverse scorpion fauna . Scorpios stings .Several epidemiological surveys have been conducted in regions affected by scorpionism -10. MostIn this study, we scrutinised the demographic and epidemiological characteristics of scorpion stings in the Touggourt region and we presented a best fit model to forecast the daily scorpion sting counts in that region using climate variables. The approach used herein has been applied in several studies related to other health issues -25, but Touggourt lies in the Sahara, in the Righ valley region of northeastern Algeria, and is flanked by sand dunes and salt lakes to the north and south and small hills to the west. It is made up of 8 districts. The region is characterised by a harsh winter and a hot and dry summer.http://www.cdc.gov/epiinfo/).Daily scorpion sting data from January 1, 2013 to August 31, 2016 were drawn from questionnaires designed to elicit information on scorpion stings from the 2 emergency service providers in the study region . The questionnaires were anonymised. The extracted data included, inter alia, the following information: sex, age, date of sting, address, anatomical sting site, sting time, time of the first medical examination, evaluation on the first clinical examination, location (inside/outside the dwelling), and treatment administered. Data were analysed and graphs were generated to recognize trends and epidemiological and demographic features of scorpion stings using the software application Epi Info version 7.2.1.0 (Daily data on the average temperature (T) and relative humidity (RH), accumulated rainfall, wind speed, and evaporation value from January 1, 2013 to August 31, 2016, was supplied by the Touggourt meteorological station.Count data occur in many fields, including public health and epidemiology. To handle count data, various models have been developed, including Poisson, zero-inflated Poisson (ZIP), Poisson hurdle (PH), negative binomial (NB), zero-inflated negative binomial (ZINB), and negative binomial hurdle (NBH) models. The Poisson model is the most widely used model for count events occurring within a specific period. A key feature of the Poisson model is equality between the mean and the variance; however, this equality does not always occur when dealing with real-world data. Indeed, real-world data are often characterised by overdispersion, which occurs when the variance exceeds the mean. When overdispersion is present, Poisson regression is usually given up in favour of NB regression, which adjusts for overdispersion. However, the NB model can only take into account overdispersion due to unobserved heterogeneity in the data, not overdispersion arising from an excess of zeroes. To handle overdispersion that results only from an excess of zeroes, zero-inflated Poisson and hurdle Poisson models are used. To account for overdispersion from an excess of zeroes and unobserved heterogeneity, zero-inflated NB or hurdle NB models are preferred given their flexibility -29. The The hurdle model is a 2-component model; a hurdle component used to model large zero counts, and a truncated count component used to model only positive counts. The hurdle model can be expressed as follows:y is the dependent variable, x is a vector of covariates for positive counts and z is a vector of covariates in the zero part. The model parameters β, γ are related to x and z, respectively, and are estimated by maximum likelihood. zerof is a probability density function at least on {0, 1} or {0, 1, 2, …}, and countf is a probability density function on {0, 1, 2, …}. The zerof part (where iy=0) is modelled using a binary logit (logistic regression) model, where all positive counts are given a value of 1. The probability of iy=0 is given bywhere zerof . Regarding countf , it is modelled with a left-truncated (iy>0) Poisson model or negative binomial model in case of overdispersion with log link. The corresponding mean regression relationship is given byThe probability of a non-zero count is therefore given by 1 - Some climate variables have been shown to affect the count of monthly scorpion stings ,13,14, sThe 6 regression models outlined in the https://www.npackd.org/p/rstudio/1.1.383) was used to perform all analyses and modelling [The RStudio version 1.1.383  cases were classified as mild, with a male-to-female ratio of 1.3, 187 (4.0%) cases as moderate, with a male-to-female ratio of 1.4, and 7 cases as severe, of which 5 occurred in females. For 147 cases (3.1%), that information was not provided. Antivenom was used in 4,455 (94.5%) patients. Seven scorpion-related human fatalities occurred . At the first medical exam, 5 of those cases were classified as severe. The victims were aged, respectively 4, 15, 26, 30, 39, 80, and 88. All of them were stung inside their dwelling and on the limbs (the lower limbs in 5 cases).Stings occur year-round, as depicted in The dataset included 1,339 observations and was divided into a training set, which included 1,095 (82.0%) observations from January 1, 2013 to December 31, 2015 to estimate the parameters of the best model, and a test set that included 244 (18.0%) observations from January 1, 2016 to August 31, 2016 to evaluate the model.2+0.19 T+9.73; R2= 0.95) and high correlation with relative humidity (r= -0.70) with a quadratic relationship ; which is consistent with results obtained in other geographical regions affected by scorpionism [An examination of the relationship between monthly scorpion sting data and the climate variables showed a strong correlation with monthly average temperature (r = 0.91) with an apparent quadratic relationship  was 4 times higher than the mean (3.52), indicating overdispersion, and close to quarter of the observations were zeroes (23.9%), regression models accounting for both overdispersion and an excess of zeroes were considered. Hence, the ZIP, PH, NB, ZINB, and NBH models were fitted to the daily scorpion sting count using the same covariates as for Poisson model. For brevity, only the NBH model estimates are presented. The estimated parameters, standard errors, and associated p-values are displayed in The zero part contains information about variables that the nested logistic regression model used to estimate the probability (ϕ) of observing a zero count:Notice that the logistic regression model did not find RH to be a useful variable for estimating ϕ. Indeed, the regression coefficient was found to be not statistically significant at the 95% CI, as indicated by the p-value (0.81) which is greater than 0.05. The 2 variables that the logistic regression model determined to be useful for estimating the probability of observing a zero count were T and Tr.The count part contains information about the variables that the NBH model used to estimate scorpion sting count on the condition that S>0.The coefficients for T and RH were statistically significant, as evidenced by their respective p-values. The coefficients for T and RH are positive, meaning that as T and RH went up, the number scorpion sting increased.By aggregating the estimated daily data for the validation period into weekly data and monthly data, the predicted data were strongly correlated with the actual data (Pearson product-moment correlation coefficient: r= 0.94 for weekly data and r= 0.98 for monthly data) as shown in The purpose of this paper was, first, to provide better insights into the demographic and epidemiological characteristics of scorpion stings in an endemic region in Algeria, and second, to model the daily scorpion sting count as a function of climate factors using 6 count data models and to compare the performance of these models.The epidemiological analysis showed a male predominance, following a similar pattern to that at the national level . SimilarIn prior studies, modelling of scorpion stings was performed with monthly data using statistical approaches such multiple linear regression, time series analysis, and count data -17. No mIn this study, for the first time, scorpion sting data were analysed without recourse to monthly aggregation as has been done in the literature to date. The fitted count regression models using the daily scorpion sting count as the dependent variable, and T, RH, and trends therein as independent variables showed that the NBH model yielded adequate fit. T and RH affected sting accidents, and T had the strongest effect. Additionally, daily predictions by means of the NBH model provided highly accurate monthly forecasts. These predictions could assist public health decision-makers in initiating appropriate, rapid, and effective measures and in containing any unusual situations.A major contribution of this study is the improvement in the forecasting accuracy of scorpion sting cases in terms of weather conditions. However, in view of the impact of scorpion envenomation in both financial and human terms, more research is needed to define alternative policies to avoid sting accidents rather than accepting them as inevitable. This novel study on the daily scorpion sting count, in terms of the statistical approach used and the results that were obtained, is a contribution towards literature and the modelling of scorpion stings that encourages further investigations and studies of scorpionism.