Purpose: To explore the efficacy and safety of lower-dose decitabine in patients with lower-risk MDS, a prospective multicenter phase II study was conducted to compare decitabine with the best supportive care (BSC).Methods: Patients diagnosed with lower-risk MDS from September 2013 to August 2018 were assigned to the decitabine group or the BSC group. Decitabine (12 mg/m2/day) was administered over 1 hour/day for 5 consecutive days in a 4-week cycle. BSC, including growth factors, transfusion, thalidomide, lenalidomide, and immunosuppressive agents were given consecutively. The endpoints included the proportion of patients who achieved overall response (OR) in the first 2 or 3 courses, event-free survival (EFS), and overall survival (OS).Results: A total of recruited 82 patients were analyzed. In the decitabine group, 65.9% (27/41) achieved OR after 2 or 3 cycles of treatment, compared with 22.0% (9/41) in the BSC group (p <0.01). Besides, 44.0% (11/25) in the decitabine group became independent of RBC/Platelets transfusion, compared with 27.8% (5/18) in the BSC group. Patients with gene mutation and treated with decitabine achieved a higher OR rate, compared with those without gene mutation . There was no significant difference in the median EFS between the decitabine and BSC groups . In the decitabine group, the most significant adverse events were infections of any grades or neutropenic fever  and one patient (4.2%) died of acute cerebral infarction within 6 weeks of treatment.Conclusion: Lower-dose decitabine demonstrated promising clinical response with acceptable toxicity profiles in patients with low- and intermediate 1-risk MDS. A higher response rate to decitabine was observed in patients with mutated genes. Therefore, lower-dose decitabine can be advocated for patients with low-risk MDS and mutated genes. Myelodysplastic syndromes (MDS) are characterized by peripheral cytopenias and dysplastic changes in bone marrow 2/day for 5 days in lower-risk MDS patients, and revealed an overall response (OR) rate of approximately 55%, with a significantly longer OS observed among those who achieved hematologic improvement (HI) 2/day) of decitabine for longer periods (5-14 days) were feasible in maintaining an advantage of its demethylating properties, which was consistent with another study 2/day for 3 days has also been shown to have a promising efficacy in lower-risk MDS patients There are several options of treatment for patients with lower-risk MDS, which include growth factors, transfusion, lenalidomide, hypomethylating agents, and immunosuppressive therapy. Decitabine represents a classic hypomethylating agent. To date, the optimal dose of decitabine in treating lower-risk patients has not been well established. Studies have examined decitabine 20 mg/meriods 5- days wer2/day) for consecutive 5 days might be optimal for patients with lower-risk MDS, and a phase 2 trial was conducted to compare low-dose decitabine with a single best supportive care (BSC) treatment.Therefore, we postulated that lower-dose decitabine . Informed consent was obtained from all patients, conforming to the institutional guidelines and in accordance with the Declaration of Helsinki.2/day intravenously over 1 hour on day 1 to day 5. The BSC treatments included administering growth factors, blood transfusion, thalidomide, lenalidomide, and immunosuppressive agents. When the patient failed to achieve any clinical response after 3 cycles of treatment, other treatment regimens would be instituted. On the other hand, responders could choose to undergo allogeneic hematopoietic stem cell transplantation , or consolidation courses, such as the previous regimen, or watch and wait. The duration of the consolidation treatment was until disease relapse, the progression of the disease, withdrawal of consent, intercurrent illness, or incidence of severe adverse events in the judgment of the investigators. Each course was administered every 4 weeks, as long as there were no significant myelosuppressive events, life-threatening complications such as severe infection, bleeding, or severe organ damage; otherwise, the interval could be interrupted, or extended up to 6-8 weeks. Blood transfusion, G-CSF, antimicrobial, and antifungal therapy were administered at the discretion of the physicians.Based on the clinical guidelines and patients' preferred option of the treatment regimen, they were assigned to either the decitabine group or the BSC group. The dosage of decitabine was 12 mg/mThe primary outcome was the OR rate, which was assessed in the first 3 cycles according to the modified International Working Group 2006 criteria SF3B1, SRSF2, U2AF1, DNMT3A, IDH1, IDH2, TET2, ASXL1, EZH2, RUNX1, ETV6, NRAS, JAK2, CBL, and TP53. Mutations were annotated using multiple databases, including 1000 Genomes, COSMIC, PolyPhen-2, and dbSNP.A target-specific next-generation sequencing (NGS) approach was used, which combined multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using an Ion Proton platform P-value of < 0.05 (2-sided) was considered statistically significant. Categorical variables were analyzed with the χ2 test or Fisher's exact test , while continuous variables were analyzed with the Student's t-test. Survival curves were constructed by the Kaplan-Meier method followed by the log-rank test. Factors associated with OR, EFS, and OS were analyzed using a stepwise backward selection approach by the logistic or COX regression.All statistical analyses were performed using SPSS 22.0 software. A A total of 88 patients across 4 treatment centers in China were enrolled in this study, with 82 patients available for data analyses Figure . Of all TET2 , U2AF1 , SF3B1 , and SRSF2 . The incidence of each gene mutation was also comparable between the two groups (Table NGS was performed in 75 patients (91.5%) before the commencement of treatment. At least 1 mutation was detected in 41 62.2%) patients. The most frequently detected mutations were .2% patieThe rates of OR and CR for the entire cohort were 43.9% and 12.2% respectively. Patients treated with decitabine had significantly higher OR  and CR  rates compared with those treated with BSC , presence of 2 or more cytopenias, good cytogenetic risk, transfusion independence, gene mutation, intermediate-1 risk disease by IPSS, and category 1 or 2 by LR-PSS were associated with a higher OR rate in the decitabine group when compared with that in the BSC group , with a total of 5 patients lost to follow-up (2 in the decitabine group and 3 in the BSC group). The EFS in the decitabine group (20.6 months) was longer than that in the BSC group (14.3 months), although this was not statistically significant (p = 0.665)  , and 46.3% (19/41) of patients developed any grade of infection or neutropenic fever. One patient treated with decitabine died of acute cerebral infarction at week-6 of treatment.2/day for 3 days) In this study, we found that lower-dose decitabine was clinically effective , and patients who were transfusion independent before treatment had a higher OR rate. Our findings were consistent with a previous study by Jabbour et al. at a lower-dose decitabine regimen . Therefore, patients with the mutated gene may benefit from early intervention with a hypomethylating agent. On the other hand, patients with mutated DNMT3A or IDH2 had been shown to have worse overall survival and more rapid progression to AML, compared to patients with wild-type DNMT3A or IDH2DNMT3A or IDH2 mutation with a shortened EFS or OS, which however were not independent prognostic factors, possibly due to the small sample size of patients harboring each of these mutations.In MDS, more than 40 recurrently mutated genes have been identified. Studies have shown that the  OR rate 2.0%. The2/day for consecutive 3 days) developed Grade 3 to 4 febrile neutropenia (25%) and infections (57%). Moreover, in lower-risk patients treated with decitabine 20 mg/m2/day for consecutive 3 days, Garcia-Manero G et al. 2/day (for consecutive 5 days) and 20 mg/m2/day decitabine (for consecutive 3 days) were comparable in lower-risk MDS patients.Several studies have shown that the incidence of adverse events is dependent on the dosage of decitabine. In the study by Lubbert M et al. In conclusion, lower-dose decitabine is effective and tolerable in patients with lower-risk MDS. Patients with gene mutations may achieve a higher response rate to decitabine than best supportive care. Further research into lower-risk MDS patients with high-risk gene mutations will shed light on how those mutations may influence the clinical response to decitabine and will provide opportunities for targeted intervention.


Postprandial hyperglycemia (PPHG) is strongly linked with the future development of cardiovascular complications in type 2 diabetes (T2D). Hence, reducing postprandial glycemic excursions is essential in T2D treatment to slow progressive deficiency of β-cell function and prevent cardiovascular complications. Most of the metabolic processes involved in PPHG, i.e., β-cell secretory function, GLP-1 secretion, insulin sensitivity, muscular glucose uptake, and hepatic glucose production, are controlled by the circadian clock and display daily oscillation. Consequently, postprandial glycemia displays diurnal variation with a higher glycemic response after meals with the same carbohydrate content, consumed at dusk compared to the morning. T2D and meal timing schedule not synchronized with the circadian clock  are associated with disrupted clock gene expression and is linked to PPHG. In contrast, greater intake in the morning  than in the evening has a resetting effect on clock gene oscillations and beneficial effects on weight loss, appetite, and reduction of PPHG, independently of total energy intake. Therefore, resetting clock gene expression through a diet intervention consisting of meal timing aligned to the circadian clock, i.e., shifting most calories and carbohydrates to the early hours of the day, is a promising therapeutic approach to improve PPHG in T2D. This review will focus on recent studies, showing how a high-energy breakfast diet (Bdiet) has resetting and synchronizing actions on circadian clock genes expression, improving glucose metabolism, postprandial glycemic excursions along with weight loss in T2D. Postprandial hyperglycemia (PPHG) in type 2 diabetes (T2D) strongly contributes to glycated hemoglobin HbA1c) values c values . FurtherThe circadian clock temporally coordinates the metabolism over a 24-h period to anticipate daily recurring feeding-fasting cycles and optimize the metabolic efficiency at an appropriate time of the day, thereby preventing metabolic dysregulation ,14,15,16The circadian clock is controlled by light/dark signals and other external inputs such as meal timing or food availability ,13,14. TMeal timing exerts a critical influence on peripheral clocks involved in postprandial glycemia ,42,43,44Circadian misalignment, with day/night cycle, often imposed in modern society, like shift workers, skipping breakfast, snacking all day, including in the evening hours, are associated with disrupted clock gene expression and linked with aberrant metabolic responses, weight gain, PPHG, increased risk for T2D ,50,51,52Asynchrony of the circadian clock is central in the pathophysiology of T2D ,13,54. LTherefore, breakfast consumption might be critical in T2D for the achievement of metabolic homeostasis and improvement of PPHG ,54,55,56In this review we discuss recent studies showing how the high energy breakfast diet designed as “big breakfast diet” (Bdiet) has resetting and synchronizing actions on clock gene expression, improving glucose metabolism, postprandial glycemic excursions, and bodyweight in T2D.The diurnal variation of the hormonal and enzymatic functions related to glucose metabolism and postprandial glycemia is synchronized by the circadian clock ,14,16.The central or “Master Clock” is found in the hypothalamus in the suprachiasmatic nucleus (SCN). The clock genes located in SCN are synchronized by light signals and generate the body endogenous ~24-h rhythm. Other clock genes are disseminated in almost all the peripheral tissues, i.e., muscle, liver, intestinal L-cells, and in β-cells ,57,58,59Noteworthy is that the first meal of the day, i.e., breakfast, exerts a more powerful resetting effect on the clock network than other meals, underscoring the damage caused by the absence or delayed breakfast on the clock regulation of glucose metabolism and PPHG ,51,57,68CLOCK (circadian locomotor output cycles protein kaput) and BMAL1 (brain and muscle ARNT-like 1) act as positive elements in the feedback loop. CLOCK:BMAL1 heterodimer drive the transcription of periods (PERs) and cryptochromes (CRYs) genes. The resulting PER and CRY proteins dimerize in the cytoplasm. After ~24 h, they are translocated back into the nucleus to interact with the CLOCK:BMAL1 complex, directly suppressing their own transcription, thus generating a cycle that recurs every ~24 h [CLOCK:BMAL1 mediates the transcription of the repressor nuclear receptor REV-ERBα and one promoter gene, the retinoic acid receptor-related orphan receptor (RORα), maintaining further the circadian (24 h) oscillation of the clock [The molecular clock mechanism is identical in central and peripheral clocks. It consists of self-sustained transcriptional-translational feedback loops ,55,58,59ry ~24 h ,55,58. Ihe clock ,58.CLOCK:BMAL1 heterodimer also mediates the transcription of tissue-specific output genes, PERs, CRYs, REV-ERBs, and RORs clock genes, along with PPARγ coactivator 1α (PGC-1α), and SIRT1 and other transcriptional elements, which promote downstream expression of several tissue-specific proteins, hormones and enzymes, relaying the clock information to cellular processes, like β-cell secretion, GLUT-4 activity, hepatic glycogenolysis, and gluconeogenesis [The ogenesis ,52,55,58BMAL1:CLOCK complex plays a critical role in the transcription of tissue-specific elements, which regulate the circadian processes involved in glucose homeostasis and postprandial glycemia [BMAL-1, RORα, and SIRT1 positively regulate the circadian β-cells secretion [BMAL-1 and RORα integrity are also necessary for circadian GLP-1 secretion of in the intestinal L-cells [REVERBα and RORα, SIRT1 and PGC-1α, modulates the gluconeogenic enzymes, and the rhythms of hepatic glycolysis pathway [glycemia ,59. BMALecretion ,20,23,60ecretion ,68, muscecretion , and β-cecretion ,60,62,69 L-cells . In the  pathway ,66,67,68BMAL1, CRY2, CRY1, and PER2, through posttranslational regulation of cAMP signaling, reduces the glucagon-stimulated hepatic glucose production [oduction , and coooduction ,66.AMPK), plays a crucial role in the clock regulation of glucose metabolism [AMPK expression significantly enhances GLUT-4 translocation and muscular glucose uptake, ensuring metabolic efficiency and improving postprandial glucose and insulin responses [AMPK exerts a positive effect on SIRT1, associated with beneficial effects on insulin sensitivity and β-cells viability [Adenosine monophosphate-activated protein kinase , and reducing energy and CH consumption in the evening hours, facilitate weight loss, improve postprandial glycemia, and reduce appetite and craving in metabolic syndrome and in T2D, compared to the inverse pattern, i.e., “high in energy and CH dinner” and reduced breakfast ,75,76,77AMPK and BMAL1, PER1 and RORα expression, and this clock gene disruption in NoB day was associated with significantly higher postprandial glycemic response and deficient and delayed insulin, and intact GLP-1 postprandial secretion after lunch [BMAL1, PER1, RORα, and AMPK [In two crossover studies in T2D patients, we explored whether skipping breakfast in a single day (NoB) versus another day consuming high energy and CH breakfast (YesB) influences the clock gene expression and the PPHG after subsequent isocaloric meals. ,56. Breaer lunch   versus breakfast consumption (YesB), was associated with significantly higher glycemia response, after lunch and also after subsequent dinner. Moreover, compared to the day when breakfast was consumed, the omission of breakfast led to reduced and delayed insulin, C-peptide, and iGLP-1 responses after lunch and dinner  Figure .The reduction of postprandial glycemia and higher and faster insulin response after lunch with prior breakfast consumption in YesB day, was previously reported in healthy and in T2D individuals ,75,78,79However, in our study this effect of breakfast was extended to dinner . Indeed,The explanation is based on a recent report showing that nutrient depletion or starvation induces lysosomal degradation of nascent insulin secretory granules and to less β-cell secretory granule biogenesis . It leadBMAL1, RORα, PER1, and AMPK [Moreover, the breakfast consumption triggers correct oscillatory clock gene expression, namely and AMPK , and theand AMPK ,56,79.AMPK, in YesB day, significantly enhances GLUT-4 translocation, muscular glucose uptake, and postprandial insulin response further reducing post meal glycemic excursions [AMPK is positively linked to SIRT1, and its beneficial effects on insulin sensitivity, β-cell proliferation and viability [The upregulation of cursions . AMPK isiability ,68,69,70Breakfast consumption is essential when targeting glycemic control in T2D. The upregulation of the clock genes induced by breakfast consumption positively influences cardiovascular activity, heart rate, blood pressure, adipose tissue, and other metabolic organs . TherefoStudies in rodents and humans suggest that not only the amount but also the hour of food intake, especially the time of energy, protein, and CH intake, play an essential role in the circadian clock regulation of energy, and glucose homeostasis, thereby influencing the glycemic postprandial excursions ,49,50,51We examined in participants with metabolic syndrome whether a diet with overall similar daily caloric intake but with different meal timing and distribution: either consuming a high-energy and CH breakfast (Bdiet) or high-energy and CH dinner (Ddiet) has a distinct influence on the glycemic postprandial response after breakfast, lunch, and dinner. We also explored the influence of Bdiet vs. Ddiet on overall glycemia, weight loss, and appetite scores. The energy distribution of Bdiet was: large breakfast , medium-sized lunch , and small dinner . In Ddiet, the plan was reversed; small breakfast and large dinner  Figure .Over 12 wk. of the study, the body weight decreased significantly in both groups. However, the Bdiet group showed a 2.5-fold more significant weight loss . After tA high-energy and CH breakfast (Bdiet) is more beneficial than a high-energy and CH dinner to reduce overall postprandial glycemia. Avoiding high energy and CH intake at dusk and in the evening may be advantageous, particularly for lowering postprandial glycemic excursions, and may reduce the risk of T2D and cardiovascular diseases.Based on the previous study , in obesCompared to the Ddiet schedule, the Bdiet led to significantly reduced overall PPHG and glucose excursions . FurtherMore recently, in insulin treated T2D, we explored the effects of either one of two isocaloric diet interventions (DI) with different meal timing and distribution during three months. One DI was aligned to the circadian clock, similar to the Bdiet of our previous studies described above, with three meals a day consisted of high energy and CH breakfast and low in energy and CH dinner (3Mdiet-Bdiet). The other DI was the traditional diet consisting of six small meals with energy and CH evenly distributed along the day, without any temporal alignment to the rhythms imposed by the circadian clock (6Mdiet).BMAL1, CRY1, PER2, RORα. Likewise, 3Mdiet-Bdiet led to an increase of daily SIRT1 levels [Compared to the traditional diet (6Mdiet), the 3Mdiet-Bdiet schedule led to a significant resetting effect in the oscillatory expression of clock genes linked to the regulation of muscular glucose uptake, hepatic glucose production and insulin secretion, namely 1 levels . This enThe design of this study doesn’t allow the direct assessment of postprandial glycemic response after each meal. However, the significantly higher glycemic excursions in 6Mdiet vs. 3Mdiet-Bdiet measured by CGM during hours assigned to meals  are highly suggestive of increased overall postprandial glycemic responses in the traditional 6Mdiet compared to 3Mdiet-Bdiet.These results are consistent with previous reports showing that misaligned meal patterns like skipping breakfast or the traditional diet consisting on frequent small meals, including snacking at night, promote asynchrony of clock gene expression and metabolic disturbances, i.e., weight gain and hyperglycemia. ,49,50,51Notably, the reduced overall glycemic excursions in 3Mdiet-Bdiet, were also significantly reduced during the nocturnal segment, suggesting an improvement in the nocturnal hepatic insulin sensitivity and reduced glucose production in 3Mdiet-Bdiet . The timImportantly the titration of total daily insulin dose 3Mdiet-Bdiet group resulted in a significant reduction in insulin requirement by 27.5 units . Also, wWe can assume in this study  that meaAs we described above, the Bdiet schedule resulted in a significant reduction in overall postprandial glycemia and weight reduction in obese non-diabetics ,40 and TIt has been reported that increased protein >35 g) intake in the breakfast leads to the reduction of all-day postprandial glycemic excursions ,86 and g g intakeIn addition to the protein load, the source and quality of the protein ingested in the breakfast are critical for its lowering effect on the glycemic postprandial response ,88. It wParticularly, Whey milk protein that accounts for 20% of whole milk protein has insulinotropic/β-cell-stimulating and glucose-lowering effects through bioactive peptides and amino acids generated during its gastrointestinal digestion ,90.These bioactive peptides can stimulate several gut hormones, including GLP-1, which stimulate β-cells insulin secretion ,93,94,95In an acute crossover study, T2D patients consumed a high glycemic index breakfast, one day preloaded with a drink containing 50 g of Whey protein and other day preloaded with water. Breakfast preloaded with Whey protein vs. water displayed a significant reduction in postprandial glucose response  Figure . FurtherThese results are in line with previous reports showing that Whey protein pre-load exerts a potent stimulatory effect on β-cell secretion, reducing postprandial glycemia in healthy ,80,93,94The increase of the postprandial GLP-1 after Whey pre-load occurred in a parallel fashion with the insulinotropic effect. This correlation supports that the higher incretin response is the mechanism subserving the more rapid and higher insulin response in the Whey protein group.Whey protein pre-load of high glycemic index breakfast stimulated the postprandial total and intact GLP-1 responses, the early prandial insulin secretion, and significantly reduced the PPHG in T2D patients. Therefore, Whey protein may represent a novel glucose-lowering strategy in T2D .We further explored in T2D patients the long-term effect of Whey protein. In this study in T2D, we tested during 12 weeks the long-term influence of Whey protein on weight loss, reduction of overall PPHG, and HbA1c. We used a well-established feeding regimen (Bdiet) consisting of a high-calorie and protein breakfast, medium-sized lunch, and low-calorie dinner ,39,56,74The participants were randomly assigned to one of the three diet intervention groups The only difference among the three diet interventions was the breakfast composition: (1) Whey protein breakfast diet (WBdiet), with high protein content at breakfast, containing Whey as the primary source of protein; (2) Protein breakfast diet (PBdiet), with high protein content at breakfast from other sources, i.e., eggs, tuna, soy; and (3) Carbohydrate breakfast diet (CBdiet), with low protein and high CH content in the breakfast . All patAlthough the three diet interventions  had similar lunch and dinner composition, the effect during a meal challenge on postprandial glycemia, insulin, GLP-1, ghrelin, glucagon, and appetite scores was not limited to breakfast but extended to subsequent meals, i.e., lunch and dinner .Compared to PBdiet and CBdiet, the Whey in the breakfast group (WBdiet) showed the lowest overall PPHG and overall AUC for postprandial plasma glucose, ghrelin, and hunger, and highest overall AUC for postprandial plasma insulin, C-peptide, intact GLP-1, and satiety scores. PBdiet showed similar benefits than WBdiet but less pronounced. WBdiet also led to a greater reduction of HbA1c and body weight compared to the other groups. The greatest reduction of PPHG was achieved after breakfast containing Whey (WBdiet) is supported by the increased GLP-1 and its insulinotropic effect, as reported in healthy and T2D individuals ,81,89,94Whey protein and its main components, i.e., alpha-lactalbumin, β-lactoglobulin, and the bioactive peptides generated during its gastrointestinal digestion, are potent secretagogues of L-cells, increasing GLP-1 intracellular levels and production ,94,95,96This study showed that in T2D individuals, a diet consisting of high-energy breakfast, medium-sized lunch, and reduced energy dinner is more beneficial in reducing overall PPHG, body weight, and HbA1c levels, when the primary protein source at breakfast is Whey, indicating that Whey protein at breakfast might be a potent adjuvant for the management of type 2 diabetes .Postprandial hyperglycemia in T2D leads to a progressive decline of β-cell function and increases the cardiovascular risk in T2D ,7. ThereAlmost all hormonal and enzymatic processes influencing the PPHG, like muscular glucose uptake, the secretion of GLP-1, and β-cell insulin production, are regulated by the circadian clock and display circadian rhythms ,22,23,24As a consequence of circadian clock regulation, the β-cell secretion and responsiveness, insulin sensitivity, and muscular glucose uptake are enhanced in the early hours of the day compared to afternoon and night ,31,32,33Meal timing, independently of the total energy intake, exerts a critical influence on peripheral clocks genes involved in regulating metabolic processes and PPHG ,45,46,47In contrast, meal-timing pattern, aligned with the circadian clock, consuming high energy and CH breakfast (Bdiet) exerts a powerful synchronizing effect on pivotal clock gene expression. It leads to a significant reduction of postprandial glycemic peaks across the day and enhanced insulin, C-peptide, and GLP-1 postprandial responses in healthy and T2D patients. Therefore, breakfast consumption is critical for achieving metabolic homeostasis and improving PPHG in T2D ,35,73,74Synchronization of the clock gene expression through a diet intervention consisting of meal timing aligned to the circadian clock by shifting more calories and CH to the early hours of the day (Bdiet) is a promising strategy for therapeutic interventions to improve PPHG, weight loss, and to prevent cardiometabolic complication in type 2 diabetes.


This retrospective cohort study compared the rates of virologically-confirmed SARS-CoV-2 infections, symptomatic or lethal COVID-19 among the residents of the Italian province of Pescara who received one or two doses of COVID-19 vaccines, versus the unvaccinated. The official data of the National Health System were used, and a total of 69,539 vaccinated adults were compared with 175,687 unvaccinated. Among the subjects who received at least one vaccine dose, 85 infections (0.12%), 18 severe and 3 lethal COVID-19 cases were recorded after an average follow-up of 38 days. Among the unvaccinated, the numbers were 6948 (4.00%), 933 (0.53%) and 241 (0.14%), respectively. The serious adverse event reports—yet unconfirmed—were 24 out of 102,394 administered doses. In a Cox model, adjusting for age, gender, and selected comorbidities, the effectiveness of either BNT162b2, ChAdOx1 nCoV-19 or mRNA-1273 vaccines was higher than 95% in preventing infections (mostly due to B.1.1.7 variant), symptomatic or lethal COVID-19. No differences were observed across genders, and among the 691 subjects who received the second dose of vaccine later than the recommended date. Although preliminary, these findings support current immunization policies and may help reducing vaccine hesitancy. Coronavirus disease-2019 (COVID-19) is spreading worldwide, and has caused over 3,200,000 deaths so far 5 May) ,5,6,7. I May ,5,6After four months of mass vaccination in Italy, given the urgency for more data to support current public health policies and potentially reduce vaccine hesitancy , we perfThis retrospective cohort study included all the subjects aged ≥18 years who were resident in the province of Pescara, Italy on 1 January 2021. The exposure was COVID-19 vaccination status , and the outcomes were SARS-CoV-2 infection , virologically-confirmed COVID-19 disease , and COVID-19-related in-hospital death.The vaccination campaign started on 2 January 2021, and Pfizer–Biontech BNT162b2, Oxford–AstraZeneca ChAdOx1 nCoV-19 and Moderna mRNA-1273 vaccines were administered gradually to the population .In order to take into account the time required for seroconversion, the start of follow-up varied depending on the administered vaccine. According to the available RCTs ,4,5, forFor the evaluation of the effectiveness of the second dose versus none, those receiving only one dose of vaccine were excluded, and the follow-up started 14 days after the second dose for all vaccines ,4,5. As For all subjects and all evaluations, the end of the follow-up was 21 May 2021 (day of the data extraction). The vaccinated subjects who received the first vaccine dose less than 14 (BNT162b2 and mRNA-1273) or 21 (ChAdOx1 nCoV-19) days before the end of follow-up were excluded from the analysis of the effectiveness of the first dose; the vaccinated subjects who received the second dose less than 14 days before the end of follow-up were excluded from the 2nd dose analysis. All unvaccinated subjects were censored at 21 May 2021.Only the outcomes that occurred after the start of the follow-up were included, and both vaccinated and unvaccinated subjects who had a positive swab or a COVID-19 disease before the start of the follow-up were thus excluded from all analyses.In order to account for some of the main potential confounders of the association between vaccination and COVID-19 or death , we usedThe Italian Ministry of Health provided the general guidelines for the National immunization campaign . Accordip-value of <0.05 was considered significant for all analyses, which were carried out using Stata, version 13.1 .First, the differences in the frequency of each recorded outcome, as well as in the other collected variables among vaccinated versus unvaccinated subjects were examined using a chi-squared test and Kruskal–Wallis test for categorical and continuous variables, respectively. For each outcome, Cox proportional hazards analysis was then used to compute the relative hazards of the subjects who received—(a) ≥1 dose; (b) 1 dose only; (c) 2 doses, versus none. All multivariate models were adjusted for age, gender, hypertension, diabetes, major cardio and cerebrovascular events, COPD, kidney diseases, and cancer, all included a priori. Schoenfeld’s test was used to assess the validity of proportional hazards assumption. A two-sided The flowchart of the study participants is shown in n = 18) with ChAdOx1 nCoV-19, and the remaining 6.1% received mRNA-1273. Among the vaccinated, 70.2% were aged 60 years or more (included in the elderly category), 8.7% were fragile individuals, 7.7% school workers, 6.9% healthcare workers, and 4.1% armed forces or civil operators.Of the vaccinated subjects, 52.8% received only one dose within the follow-up; 68.5% received BNT162b2 vaccine; 24.4% ChAdOx1 nCoV-19, and 7.0% mRNA-1273. Among those who received the second dose, 93.8% were vaccinated with BNT162b2, only 0.1% .p < 0.001; p < 0.001). A total of 244 SARS-CoV-2-positive persons died—241 (0.14%) were not immunized (seven of whom younger than 50 years); three received at least one dose of vaccine .Overall, 7033 SARS-CoV-2 infections have been recorded during the follow-up—6948 (2.87%) among the unvaccinated, and 85 (0.12%) among those who received at least one dose (n = 0 infections); and 95% lower for those who received one or two doses of any vaccine . The effectiveness after a single dose varied by vaccine, ranging from 55% (BNT162b2) to 95% . In the sample restricted to those who received only one vaccine dose, the COVID-19 cases were 18 (0.11%), 0 and 0 among those vaccinated with BNT162b2, ChAdOx1 nCoV-19 and mRNA-1273, respectively . Vaccine effectiveness to prevent death was very similar—98% to 100% after two doses of either BNT162b2 or mRNA-1273, with no deaths among those who received a single dose of ChAdOx1 nCoV-19 or mRNA-1273, and two deaths among those who received only one dose of BNT162b2.While the average follow-up of those receiving a single dose of ChAdOx1 nCoV-19 was 30.4 days, the mean follow-up duration of the subjects who received only one dose of m-RNA vaccines was too short  to permit a meaningful evaluation of the effectiveness of these vaccines against outcomes, such as COVID-19 or death, that may require 3–20 days to occur after the contagion .All analyses were repeated separately by gender —after twAs an additional secondary analysis, we stratified the subjects who received two doses of vaccine according to the number of days between the first and second dose—those who received the second dose within the time interval recommended by the producers  were coFor a total of 102,394 administered doses, the overall number of serious adverse event reports that were received during the follow-up was 24 . The regional network is currently evaluating these reports in order to verify how many of them could have been caused by the vaccination.In the general population of an Italian province, where 28.4% of the citizens received at least one dose of mRNA (BNT162b2 or mRNA-1273) or adenoviral-vectored (ChAdOx1 nCoV-19) COVID-19 vaccines, and were followed for an average of 38 days after seroconversion, the proportion of virologically confirmed SARS-CoV-2 B.1.1.7 variant infection, COVID-19 symptomatic disease, and COVID-19-related death were drastically lower among vaccinated versus unvaccinated individuals.Overall, adjusting for age, gender, selected comorbidities, and follow-up duration, the risk of infection decreased by 98% after two doses of vaccine, and by 55% to 95% after one dose, depending on the vaccine type. Vaccine effectiveness was even higher against COVID-19 severe or lethal diseases, which were reduced by 99% and 98%, respectively, after two doses of BNT162b2 or mRNA-1273, or one dose of ChAdOx1 nCoV-19.These findings are consistent with those of recent observational studies on vaccine protection against severe COVID-19, in the context of mass vaccination campaigns, that reported an effectiveness ranging from 57% to >90% after one dose, and ≥90% after two doses ,14,20,21Although higher than what is reported by preliminary CDC data 0.5 × 10,000) , the rat × 10,000No significant differences were observed in vaccine effectiveness across genders at any dose and for any outcome. This finding is in line with previous trials and real-world studies on healthcare workers ,4,14, ann = 691), as compared to people vaccinated according the standard regimen [In the current scenario of limited vaccine availability, some EU countries, including Italy, have decided to delay the administration of the second dose of BNT162b2 vaccine (from three to six weeks after the first dose), in order to use all available doses to expand the proportion of population who received at least one dose of vaccine ,32. Whil regimen . These f regimen , in whicThe current pandemic scenario is further complicated by the presence of new SARS-CoV-2 variants. Concerns were raised about the efficacy of currently available vaccines, and the durability of the immune response, against the three currently prevalent variants . By now,Taken together, these findings suggest that COVID-19 vaccine benefits largely outweigh their risks. Our results are in line with the available data; however, the duration of follow-up is limited, and validated data on vaccine-linked serious adverse events are still being awaited. Also, although our analyses relied upon a dataset including vaccination and clinical data extracted from routinely collected electronic health records, observational studies are prone to recall or misclassification bias , and biaWith regard to the latter concept, previous research suggested that some of the key drivers of vaccination uptake are the perceived efficacy of a vaccine, and concerns over serious adverse events . This wa


A high percentage of patients who undergo surgical resection for hepatocellular carcinoma (HCC) experience recurrence. Therefore, identification of accurate molecular markers for predicting recurrence of HCC is important. We analyzed recurrence and non-recurrence HCC tissues using two public omics datasets comprising microarray and RNA-sequencing and found novel gene signatures associated with recurrent HCC. These molecules might be used to not only predict for recurrence of HCC but also act as potential prognostic indicators for patients with HCC.CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were identified, and the expression of these genes was evaluated using quantitative reverse transcription PCR in the validation set (n = 57). The genes CETN2, HMGA1, RACGAP1, and SNRPB, but not MPZL1, were upregulated in patients with recurrent HCC. In addition, the combination of HMGA1 and MPZL1 demonstrated the best area under the curve  for predicting HCC recurrence. In terms of clinicopathological correlation, CETN2, MPZL1, RACGAP1, and SNRPB were upregulated in patients with microvascular invasion, and the expression of MPZL1 and SNRPB was increased in proportion to the Edmonson tumor differentiation grade. Additionally, overexpression of CETN2, HMGA1, and RACGAP1 correlated with poor overall survival (OS) and disease-free survival (DFS) in the validation set. Finally, Cox regression analysis showed that the expression of serum alpha-fetoprotein and RACGAP1 significantly affected OS, whereas platelet count, microvascular invasion, and HMGA1 expression significantly affected DFS. In conclusion, HMGA1 and RACGAP1 may be potential prognostic biomarkers for predicting the recurrence of HCC after surgical resection.Hepatocellular carcinoma (HCC) has a high rate of cancer recurrence (up to 70%) in patients who undergo surgical resection. We investigated prognostic gene signatures for predicting HCC recurrence using in silico gene expression analysis. Recurrence-associated gene candidates were chosen by a comparative analysis of gene expression profiles from two independent whole-transcriptome datasets in patients with HCC who underwent surgical resection. Five promising candidate genes,  Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related mortality worldwide . ImmunotNUF2 is related to the recurrence of HCC, and that higher NUF2 expression is associated with poor overall survival (OS) and recurrence-free survival in patients with HCC , 0.812 for HMGA1 (95% CI: 0.728–0.879), 0.731 for MPZL1 (95% CI: 0.639–0.809), 0.890 for RACGAP1 (95% CI: 0.818–0.941), and 0.765 for SNRPB (95% CI: 0.676–0.839).The validation set included 57 matched pairs of human HCC tissues and adjacent non-tumor tissues. The baseline characteristics are shown in MPZL1, the genes were significantly upregulated in patients with recurrent HCC compared to expression levels in patients with non-recurrent HCC. Next, we evaluated the prognostic performance of the five selected genes in recurrent HCC compared to serum alpha-fectoprotein (AFP) using AUROC. The AUC of serum AFP for predicting HCC recurrence was 0.628, and the AUC of CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were 0.689, 0.755, 0.524, 0.754, and 0.677, respectively. No single gene was found to be superior to serum AFP for predicting tumor recurrence   D; right.CETN2, MPZL1, RACGAP1, and SNRPB were significantly overexpressed in patients with vascular invasion, but HMGA1 was not overexpressed , creatinine (p = 0.043), serum alanine aminotransferase (ALT) (p = 0.035), serum AFP (p < 0.001), protein induced by vitamin K absence-II (PIVKA II) (p = 0.003), advanced tumor stage , SNRPB expression (p = 0.044), CETN2 expression (p = 0.020), HMGA1 expression (p = 0.020), and RACGAP1 expression (p < 0.001) were significantly associated with OS. In addition, platelet (p = 0.015), serum AFP (p = 0.003), PIVKA II (p = 0.002), presence of microvascular invasion , advanced tumor stage , SNRPB expression (p = 0.008), CETN2 expression (p = 0.008), HMGA1 expression (p = 0.001), and RACGAP1 expression (p < 0.001) were significantly associated with DFS  and RACGAP1 expression . The prognostic factors for DFS were platelet , presence of MVI , and HMGA1 expression   .The present study compared gene expression between recurrent HCC tissues and non-recurrent HCC tissues after surgical resection with integrative analysis using two different datasets (GSE89377 and GSE114564) analyzed by microarray and RNA-seq, respectively. Microarray and RNA-seq are two of the most commonly used high-throughput technologies for gene expression profiling. However, little is known about the reproducibility of these results. Therefore, we calculated the correlation between microarray and RNA-seq results to explore the reproducibility between the two techniques and to find more precise markers.CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB) associated with HCC prognosis that are potential biomarkers for HCC recurrence. Among the five genes, CETN2, HMGA1, RACGAP1, and SNRPB were significantly upregulated in patients with recurrent HCC. Additionally, the combination of HMGA1 and MPZL1 had the best AUC for predicting HCC recurrence.We performed GSEA with four different gene sets that were previously studied for HCC recurrence to identify the relevance of the verified genes with recurrence. For more accurate verification, we used the GSE39791 database to evaluate the expression of 19 genes to confirm that they were upregulated in HCC tissues. We identified five genes .Located on chromosome 6p21, d HMGA1c . Althougelopment ,23. Receg breast ,25, lungg breast ,27, and g breast ,29 cancet of HCC ,31, only for HCC . In the A member of the immunoglobulin superfamily, MPZL1 is composed of an extracellular segment and an intracellular portion with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) [MPZL1 is overexpressed in malignant tumor tissues [MPZL1 expression and its relationship with HCC prognosis and recurrence have not been evaluated. This study is the first to report that an increased expression of MPZL1 is associated with poor prognosis of HCC. According to the Edmondson grade, the expression of MPZL1 was increased in patients with invasion although MPZL1 expression was not significantly different between recurrent HCC patients and non-recurrent HCC patients. These results support the view that the combination of HMGA1 and MPZL1 may be a potential biomarker as they act in a complementary way, predicting recurrent HCC with an AUC of 0.807 (95% CI = 0.681–0.899). (ITIMs) . The pho (ITIMs) ,35. Alth tissues ,37,38,39RACGAP1 upregulation in HCC: ECT2 interacts and co-localizes with RACGAP1 and protects RACGAP1 from degradation; and, RACGAP1 promotes ECT2-mediated RhoA activation and metastasis in HCC cells [RACGAP1P activates RACGAP1, and miR-15-5p suppresses the expression of both RACGAP1P and RACGAP1 [MAGI2-AS3 interacts with KDM1A and promotes histone demethylation of histone H3 lysine 4 (H3K4me2) in the RACGAP1 promoter region, and silencing RACGAP1 inhibits tumor growth in vivo [RACGAP1 is associated with early recurrence of HCC [RACGAP1 expression could be an independent prognostic factor for HCC by Cox proportional hazard regression analysis .A number of studies have identified the underlying mechanisms of CC cells . Wang et RACGAP1 . The lnc in vivo . Regardie of HCC . In the SNRPB has been reported to be associated with poor prognosis in patients with HCC. It has also been suggested that c-Myc mediates upregulation of SNRPB [Overexpression of of SNRPB . The SNRof SNRPB . Our stuIt remains unclear whether expression of specific genes in HCC tissue could be an independent predictor of recurrence. To identify recurrence-related genes in HCC, we proposed a detailed methodology through combinatorial analysis between two different platforms, microarrays, and RNA-seq datasets. As a result, CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were identified as potential predictive biomarkers of recurrent HCC. Indeed, we have demonstrated that the combination of HMGA1 and MPZL1 has excellent prognostic value for distinguishing patients at high risk of HCC recurrence in the validation cohort. These patients at high risk of recurrence may achieve better clinical outcome by active change of their post-treatment surveillance intervals and/or the adjuvant treatment to prevent recurrence. In the future, more accurate clinical risk prediction model for HCC patients at high risk of recurrence would be derived by integrating the expression of recurrence-related genes and various clinical characteristics; and, the present study would serve as a cornerstone for deriving the more accurate risk prediction model for HCC patients.HMGA1, MPZL1, RACGAP1, and SNRPB in cancer progression were reported in several prior studies. However, the mechanism of CETN2 action in cancers, specifically HCC, was not identified. Further studies are required to evaluate the underlying mechanism of CETN2 in patients with HCC.This study has some limitations. First, the number of patients enrolled in the validation study was relatively small. Besides, the validation study was designed as a retrospective study, and it was not performed in an external cohort. Thus, to integrate our results into real clinical practice to discriminate patients at high risk of recurrence, rigorous external validation in a larger prospective cohort should be performed. Second, we did not perform the mechanism studies underlying the promotion of HCC recurrence by the recurrence related genes. Among the five genes, the mechanism of CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB. Furthermore, the combination of HMGA1 and MPZL1 showed excellent predictive capabilities for HCC recurrence. Moreover, HMGA1 and RACGAP1 could be used as independent prognostic indicators in patients with HCC. The integrative analysis that we used in this study could be used to derive genes that could be used as potential biomarkers for HCC recurrence, and these genes may help identify patients who need to be followed closely to lower the recurrence rate after liver resection.In conclusion, the present study derived potential molecular biomarkers for recurrent HCC using a systematic, genome-wide discovery approach. The following were significantly overexpressed in patients with HCC and were significantly correlated with HCC recurrence:


Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna turionifera, Lemna minor, and Wolffia globosa. All six duckweed species preferred S. polyrhiza. The expression of genes encoding nitrate reductase and nitrite reductase increased about 10-fold when GS1;2 and the GS2 by 2- to 5-fold, respectively, but repressed GS1;1 and GS1;3. cis-elements, TATA-based enhancers, Duckweed plants play important roles in aquatic ecosystems worldwide. They rapidly accumulate biomass and have potential uses in bioremediation of water polluted by fertilizer runoff or other chemicals. Here we studied the assimilation of two major sources of inorganic nitrogen, nitrate ( The application of nitrogen (N) fertilizers produced substantial crop yield increases, but N fertilizers also cause serious environmental problems . Plants monium NH+) 4]. A (NH4+)) . Water e (NH4+)) . The res. A , represented by 37 worldwide distributed species ,13,14, hArabidopsis thaliana and rice (Oryza sativa) [Plant biomass accumulation is strongly associated with N utilization, and duckweed plants are extremely efficient at assimilating N. For example, duckweed nitrogen use efficiency (NUE) reached more than 68 kg biomass/kg N under N limitation due to N remobilization and recycling by the ubiquitin-proteasome system and autophagy . However sativa) .N mostly enters into plant tissues in inorganic form (s (AMTs) ,30. Inorlutamate .Higher plants contain several GS isoenzymes, which are located in the cytosol (GS1) and in the plastids (GS2) and are encoded by a small multigene family . CytosolLandoltia punctata) [One important aspect of N assimilation is the plant’s preference for rce of N , a questrce of N ,29. Mosthe plant . Moreove species ,35. Howe species , demonstunctata) , which i+ stress .Spirodela (S. polyrhiza), Landoltia (L. punctata), Lemna , and Wolffia (W. globosa). Taking advantage of the available genome sequence of great duckweed (S. polyrhiza) [S. polyrhiza grown in media supplied with Here, we explored the utilization of lyrhiza) , we charSpirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, L. turionifera, and Wolffia globosa) and Lemna minor, collected in Kazakhstan. Prior to the N assimilation experiments, the identity of all species grown in vitro from a single frond was confirmed by barcoding through sequencing the atpF–atpH (ATP) and psbK–psbL (PSB) intergenic spacers [The duckweeds used in this study include five species isolated in Eastern China , exhausted the N in the medium by day 8 independent of the source, with S. polyrhiza, L. punctata, and L. turionifera showing the most rapid consumption.We observed almost identical dynamics of N consumption by the duckweed species grown for 12 days in medium containing 5 mM of N source . Five spS. polyrhiza, L. punctata, and L. turionifera) consuming almost all of the available When grown in medium supplied with both tivation . The ducS. polyrhiza as the representative species due to the availability of a well-characterized whole-genome sequence [GS genes, NR, NiR, NADH-GOGAT and SpFd-GOGAT for the S. polyrhiza ecotype NB5548 used in this study .We evaluated the key N assimilation genes encoding NR, NiR, GS, NADH-GOGAT, and Fd-GOGAT, which have been identified as the major players in the assimilation of inorganic N in many plant species , using Ssequence ,40,41,42S. polyrhiza ecotype Sp9509 genome [BLAST searches of the 9 genome , availabS. polyrhiza. For comparison, most terrestrial diploid plants have 2–3 NR genes and one NiR gene. SpNR is composed of four exons and three introns, typical for this plant lineage, whereas SpNiR has three exons and two introns  are a family of enzymes involved in the primary incorporation of inorganic N in the form of  for GS2 ,44.GS genes, searched from the two available S. polyrhiza genomes using the rice protein sequence of OsGS1;2 as a query, and showing no sequence variability between S. polyrhiza ecotypes 9509 and 7498, were classified as SpGS1;1, SpGS1;2, SpGS1;3, and SpGS2 based on sequence similarities with the corresponding GS genes from rice, barley (Hordeum vulgare), and sorghum. All analyzed duckweed GS genes were composed of 13 exons and 12 introns, with no size variation of exons 1 through 12  for SpGS1;1-1;3 but some variation in the intron lengths. Compared to the three SpGS1 genes, SpGS2 is a bit larger in size due to longer introns, and longer exons 1 and 13, which contain a chloroplast signal peptide and short variable C-terminal extension peptides , an aquatic dicot plant.Alignments of the amino acid sequences deduced from genomic DNA sequences of European and American ecotypes 9509 and 7894 and cDNA of Chinese ecotype NB5548, showed a very high degree of similarity between GSs in duckweed and other plants representing both monocot and dicot species . The phyS. polyrhiza possesses a single gene for Fd-GOGAT and one for NADH-GOGAT  [Fd-GOGAT genes in many species; for example, lotus NnFd-GOGAT has 33 exons reaching almost 200 kb in size, while the Fd-GOGAT genes usually are of more than 330 kb in conifers [GOGATs and GS2 form a GS/GOGAT cycle in plant chloroplasts, where GS catalyzes the formation of Gln from Glu and s of Glu . The genDH-GOGAT . Similarestivum)  and riceestivum) , containconifers .S. polyrhiza grouped with GOGAT proteins from dicots.Mature GOGAT proteins demonstrate high sequence conservation . AccordiS. polyrhiza samples for RNA isolation were taken simultaneously with medium sampling for measurement of N as represented in β-actin and histone H3), and related to the gene expression levels at the starting starvation point (day 0)  .NR and NiR was strongly induced in duckweed cultivated in medium with NR and NiR, respectively. The expression of NR and NiR then decreased slightly at day 4 following the drop in available NR and NiR decreased about 2.5- and 1.5-fold on day 2, respectively, then the expression of both genes increased about 1.5-fold on day 4, indicating the start of their induction. Moreover, the relative expression of NR and NiR similarly decreased about 7- and 10-fold on day 2 and day 4, respectively, when The expression of GS1;2 and GS2 gradually increased, with a more pronounced increase in samples grown in the presence of GS1;1 and GS1;3 was suppressed by the addition of The relative expression of Fd-GOGAT and NADH-GOGAT were induced by either Fd-GOGAT increased 1.6- and 2.1-fold on day 2 and day 4, respectively, when 5 mM NADH-GOGAT increased 11.6- and 10.9-fold on day 2 and day 4, respectively, when 5 mM The relative expression of S. polyrhiza, we analyzed the gene promoter regions for the presence of possible regulatory cis-elements. The survey revealed the presence of cis-elements similar to the nitrate-responsive elements (NRE), first described for the NiR promoter in A. thaliana [SpNR, SpNiR, SpGS1.1, SpGS1.2, SpGS1.3, SpGS2, SpNADH-GOGAT, and SpFd-GOGAT showed some divergence from the canonical A. thaliana bipartite pseudo-palindromic sequence GACcCTT-N(10)-AAGagtcc, most of them aligned relatively well with the corresponding NREs found in A. thaliana, rice, sorghum, and maize (Zea mays)  .SpNiR harbors four NRE-like copies positioned within the 242-bp promoter region upstream of the translation start site; SpGS1.2 and SpNADH-GOGAT, which along with SpNiR demonstrated the highest upregulation by S. polyrhiza genes, possess three NRE-like elements each , SpGS1.2 (6), SpGS2 (4), and SpNADH-GOGAT (2), representing a potential opportunity for negative regulation by the SpNIGT1 homolog upon supply of cis-elements were found in the promoters of SpNiR and SpGS1.3, while SpNR and SpFd-GOGAT both contain a single copy of GAATC/GATTC  family proteins, first identified as transcriptional repressors in rice , and latthaliana ,60. NIGTGAATATTC ,61. A seTC/GATTC .SpGS1.1, contained repetitive GAGA or CTCT stretches between −1070 and −330 nucleotides upstream of the ATG start site, with the SpFd-GOGAT promoter containing an exceptionally long GAGA region of 306 bp (positions −864 to −559) and a 22-nucleotide stretch of TCTC at −351 to −329 bp  are secondary nucleotide structures found in guanidine-rich regions, which are implicated in various cellular processes in eukaryotic organisms . The G4 ine tool  preset fine tool  revealedart site . The proas maize , rice [7as maize , wheat [as maize , and baras maize .Lemna gibba [Landoltia punctata [The duckweed species investigated in this study  are the na gibba  and Landpunctata , which a+ stress . In our xhausted .From the standpoint of cell metabolic economics, f growth ,35. Duck+ stress , but itsSpNR and SpNiR demonstrated very similar, almost identical, expression patterns in response to SpNR and SpNiR expression, especially when it was supplied as the sole N source at a relatively high concentration of 5 mM. On the one hand, the stimulation of NR and NiR by Lemna species [In accordance with their functional link in the step-by-step conversion of and NO3− . The exp species ,78. On t species , barley  species ,81, and  species .NR and NiR  [GS1;2 and GS2 likely play a central role in the plant’s high growth rate and biomass accumulation. tissues ,88, espe tissues  and rice tissues , where ipiration . These ginensis) . Moreoveinensis) ,92. In dSpGS1;2 and SpGS2, addition of fresh nutrient medium to an N-starved duckweed culture  [HvGS1;1) [Brassica napus; BnaGLN1.1 and BnaGLN1.4) [OsGS1;1 showed a severe reduction in grain yield [TaGS1.1 knocked down by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) had reduced N translocation efficiency and grain filling, fewer grains per spike, and significantly reduced yield compared to the wild type [HvGS1;1 led to higher grain yields and NUE in barley [ZmGln1;3 in maize resulted in an increase in kernel number, ultimately leading to a higher yield in transgenic plants compared with controls [In contrast to nescence ,94. The TaGS1.1) , barley HvGS1;1) , and oilaGLN1.4)  are all aGLN1.4) ,89,96,97ild type . To the n barley . Similarcontrols .SpGS1;1 and SpGS1;3 to primary N assimilation by duckweed is much lower than that of SpGS1;2 and SpGS2. Accordingly, we suggest that SpGS1;1 and SpGS1;3 may play a role in filling of the turion with storage nutrients. Preliminary support for this assumption can be found in RNA-seq data related to turion formation in S. polyrhiza [SpGS1;3 and simultaneous 5-fold downregulation of SpGS1;2.Taking these observations together, it might be concluded that, similar to other plants , the conolyrhiza , showingSpNADH-GOGAT and SpFd-GOGAT expression by N added after starvation, where the relative expression of Fd-GOGAT increased about 2-fold, and the relative expression of NADH-GOGAT increased by more than 10-fold. While the GOGAT isozymes are both implicated in N assimilation processes, such as primary assimilation and in photorespiration [Approximately 95% of the H-GOGAT) ,104. Ourpiration ,106, rempiration ,107, andpiration ,108, ourS. polyrhiza promoters. While some of these motifs show noticeable divergence from the canonic bipartite NRE, especially the 5′-halves of the NREs in the SpNADH-GOGAT promoter  was first discovered and thoroughly characterized in Drosophila, where GAFs regulate numerous developmental genes in cooperation with chromatin remodeling factors [Spirodela species, S. polyrhiza and S. intermedia, are unusually enriched in (GA/CT)n repeats as documented by whole-genome surveys [SpGS1;1, where a strong G4 structure was identified in the antisense strand of the first intron. Therefore, our analysis identified significant enrichment of G4 structures primarily in the promoter regions of the duckweed genes involved in N assimilation.The binding of (GA/CT) factors ,110. The surveys ,111. How surveys ,112, the surveys . Also, i surveys . No suchS. polyrhiza promoters n repeats, and G-quadruplex structures, while the details of their individual roles and interaction will need to be uncovered in future research.Overall, the array of potential regulatory DNA elements revealed in the S. polyrhiza (collection ID: NB5548), L. punctata (NB0031), L. turionifera (NB0013), and W. globosa (NB0015)) were collected from small ponds and lakes at different locations between Huai’an city and Hongze lake in eastern China; L. aequinoctialis (NB0007) originated from the lake in a park next to People’s Square in Shanghai; and L. minor (NB0020) was sampled in Nursultan, Kazakhstan. To propagate the samples under sterile conditions and form a stock of live material for further experiments, the collected duckweed samples were surface sterilized in a solution containing 0.5% sodium hypochlorite and 0.1% benzalkonium bromide, washed with autoclaved water, and single fronds were put on solid agar medium supplemented with SH salts [atpF–atpH (ATP) and psbK–psbL (PSB) following the protocol described by Borisjuk et al. (2015) [The ecotypes used in this study were selected from the duckweed live in vitro collection recently established in the School of Life Sciences at Huaiyin Normal University, Huai’an, China  .−2 s−1 provided by cool white fluorescent bulbs in a 16-h light/8-h dark cycle. After four weeks of growth, the accumulated fronds were weighed and 0.5-g portions were inoculated into open 500-mL paper containers containing 200 mL of basic SH medium (no sugar) supplemented with three different formulations of N. The first medium was supplemented with To accumulate biomass, duckweed plants grown on solid agar medium were initially transferred into sterile liquid SH medium supplemented with 5 g/L sucrose and cultivated at 23 ± 1 °C with a photon flux density of 50–60 μmol·mThe total nitrogen (TN) concentration was determined using standard alkaline potassium persulfate digestion followed by UV spectrophotometry as previously described . The NO3d 275 nm . The NH4r method .To access the duckweed orthologues of genes encoding critical enzymes involved in N assimilation, i.e., nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), NADH-dependent glutamate synthase , and ferredoxin-dependent glutamate synthase , we searched the available duckweed genome sequences using the reference rice protein sequences OsNR (XP_015622710), OsNiR (XP_015641702), OsGS1;2 (XP_015631679.1), OsNADH-GOGAT (XP_015649242.1), and OsFd-GOGAT (XP_015646712.1) as the initial queries in tBLASTn searches.S. polyrhiza, ecotype NB5548, mRNA as a template. The PCR fragments were amplified with gene-specific primers designed according to the in silico sequence information available at NCBI  for S. polyrhiza, ecotype 9509 [The genes coding for SpNR, SpNiR, SpGS1;1, SpGS1;2, SpGS1;3, SpGS2, SpFd-GOGAT and SpNADH-GOGAT were validated by sequencing the PCR-amplified gene regions using cDNA prepared from local ype 9509 , cloned A. thaliana, lotus (Nelumbo nucifera), and sorghum (Sorghum bicolor), available in GenBank [The intron/exon structure of the duckweed genes was deduced based on similarities with the homologous genes of rice,  GenBank , followi GenBank .Mature protein sequences were compared with a selection of monocot and dicot sequences available in public databases ,119,120.NR, NiR, GS1;1, GS1;2, GS1;3, GS2, Fd-GOGAT, and NADH-GOGAT) were measured using reverse transcription quantitative PCR (RT-qPCR) with gene-specific primers designed based on the deduced exon sequences  [The transcript levels of eight target genes (equences . For gen. (2011) . The qua−ΔΔCt method [S. polyrhiza ecotype NB5548 housekeeping genes, histone H3 and β-actin , according to the geNorm protocol [The qPCR reactions were performed using CFX Connect Real-Time detection system  using the UltraSybr Mixture (High Rox) supplied by CWBio . The cycling conditions were as follows: initial denaturation at 95 °C for 10 min followed by 40 cycles of 30 s at 94 °C, and 20 s at the annealing temperature of the respective primers. The SYBR Green I fluorescence was monitored consecutively after the annealing step. The quality of products was checked by a thermal denaturation cycle. Only results providing a single peak were considered. The coefficient amplification efficiency for each pair of primers was determined by 10-fold serial dilutions. The level of relative expression was calculated by the 2t method . Expressprotocol . Three rThe present study demonstrated that duckweeds efficiently assimilate The obtained data also provides valuable information for improvement of nitrogen assimilation efficiency, NUE, and phytoremediation of wastewater, potentially by up- or down-regulating certain genes , modulat


Penicillium contains over 480 species of fungi, and their initial identification is predominantly based on their macroscopic colony morphologies and light microscopic characteristics (–BenA) and calmodulin (CaM), Penicillium species are not easily identified. Mycologists have known for years that there are many inaccurate labels in GenBank for the genus Penicillium and other fungal groups , RPB1, RPB2, small subunit (SSU), and Tsr1, should be included in the classification of Penicillium species  and limited the query to sequences from type material. With this framework, here we are renaming the three Penicillium strains according to the updated BLASTn results.In 2015 to 2017, using colony morphology, microscopic characteristics, and the ITS barcode, we identified two orum 113 . At thatrum 113 79. More rrum 113 7, we repePenicillium strain NJ1, BenA BLASTn results indicated that the top hits were Penicillium crustosum strains NRRL 66388, DI16-101, and CBS115503; among them, CBS115503 is the ex-type strain. The CaM BLASTn search for strain NJ1 indicated that the three top hits were P. crustosum strains KrP/6, DTO266-B3, and imi91917 (the ex-type strain). Their maximum/total scores, query coverage, E values, percent identity, and accession numbers are listed in Penicillium NJ1 strain that we called P. solitum should be renamed P. crustosum NJ1.For Penicillium strain RS1 BenA BLASTn results indicated that the top hit was the Penicillium polonicum ex-type strain CBS222.28, and BLASTn results for the CaM sequence of this strain indicated that the three top hits were P. polonicum strains F775, CMV001E2, and CBS222.28 (the ex-type strain). Therefore, this strain should be renamed P. polonicum RS1. Finally, the BLASTn results for the BenA and CaM sequences of strain 113, which was isolated from a flooded home, indicated that the top hits were Penicillium maximae strains SFC20151014-M14, NRRL2060 (the ex-type strain), and SL-CL7. Therefore, strain 113 should be renamed P. maximae 113. Phylogenetic analyses using genome data from strains NJ1 and RS1 also support new names.Similarly, the Penicillium (Molecular identification is the default approach for the identification of microfungi . With thicillium , 12.


Among pregnant women, the most frequent RAMs were K103N , M184V , Y181C/V/I , P225H , and K219N/E/Q/R . Protease and integrase inhibitor-RAMs were relatively few or absent. Based on the genotype susceptibility score distributions, 73%, 88%, and 14% of children, adolescents, and pregnant women, respectively, were not susceptible to all three drug components of the WHO preferred first-line regimens per 2018 guidelines. These findings suggest that routine HIVDR surveillance and access to better ART choices may improve treatment outcomes in Sierra Leone.Human immunodeficiency virus (HIV) drug resistance (HIVDR) is widespread in sub-Saharan Africa. Children and pregnant women are particularly vulnerable, and laboratory testing capacity remains limited. We, therefore, used a cross-sectional design and convenience sampling to characterize HIV subtypes and resistance-associated mutations (RAMs) in these groups in Sierra Leone. In total, 96 children , 47 adolescents , and 54 pregnant women  were enrolled. Median treatment durations were 36, 84, and 3 months, respectively, while the sequencing success rates were 45%, 70%, and 59%, respectively, among children, adolescents, and pregnant women. Overall, the predominant HIV-1 subtype was CRF02_AG , with minority variants constituting 12%. Among children and adolescents, the most common RAMs were M184V (76.6%,  According to the World Health Organization (WHO) 2019 report on HIV drug resistance (HIVDR), the emergence and spread of HIVDR in low- and middle-income countries (LMICs) was a major factor limiting the success of antiretroviral treatment (ART) programs in achieving the 90–90–90 global targets for 2020 [Sierra Leone is a low-income West African country of 7.4 million people. The HIV epidemic in the country is described as generalized, with an estimated national HIV prevalence of 1.5% in 2018, i.e., 70,000 people living with HIV, of whom 6600 were children aged 0–14 years . The NatDespite the welcome improvements in the areas of HIV care outlined above, barriers persist in other areas of the national AIDS response in Sierra Leone. For example, the UNAIDS 2019 report cited earlier observed that there was uneven progress across the 90-90-90 targets. In 2018, fewer than half (49%) of PLWH in Sierra Leone knew their HIV status, and 41% of HIV-diagnosed patients were receiving ART, while only 26% of those on treatment were virally suppressed, the latter being likely due to a combination of poor treatment adherence and high prevalence of HIVDR in this setting . In addin = 64) and an acquired HIVDR (ADR) prevalence >95% (n = 151) among adult HIV-infected individuals aged >18 years who were receiving routine HIV care at Connaught Hospital, the country’s main referral health facility in Freetown [In the first study characterizing HIVDR in Sierra Leone, we previously observed a pre-treatment HIVDR (PDR) prevalence of 37%  children aged 2–9 years inclusive, (2) adolescents aged 10–18 years inclusive, and (3) pregnant women aged >18 years. The children and adolescents were enrolled at the HIV Clinic at Ola during Children’s Hospital, while the pregnant women were recruited at the HIV and Antenatal Clinic at the Princess Christian Maternity Hospital located in Freetown, Sierra Leone. Both hospitals serve as the national referral health centers for obstetric and pediatric cases and are major teaching affiliates of the College of Medicine and Allied Health Sciences of the University of Sierra Leone. The children and pregnant women were not biologically related.Due to lack of HIVDR testing capability in Sierra Leone, none of the study participants had ever had baseline HIVDR testing at ART initiation; study participants were, therefore, selected consecutively using convenience sampling. At the time of study enrollment, the Sierra Leone National HIV Treatment Guidelines from 2017  recommended a dual nucleos(t)ide reverse transcriptase inhibitor (NRTI) backbone, i.e., tenofovir/lamivudine (TDF/3TC), abacavir/lamivudine (ABC/3TC), or zidovudine/lamivudine (AZT/3TC) for plus the non-nucleotide reverse transcriptase inhibitors (NNRTIs) efavirenz (EFV) or nevirapine (NVP) as the preferred first-line regimen for all age groups. The ritonavir-boosted protease inhibitors (PIs) lopinavir (LPV/r) or atazanavir (ATV/r) were recommended as preferred second-line treatment for adults, or as alternative first-line for children <3 years who were exposed to nevirapine for prevention of MTCT.The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Case Western Reserve University/University Hospitals Cleveland Medical Center (approved 16 July 2019) and the Sierra Leone Ethics Scientific and Research Committee (approved 28 February 2019). Written informed consent was obtained from participants aged ≥15 years, whereas parental or guardian consent was obtained for participants <15 years.® K2 EDTA, Waltham, MA, USA) and centrifuged at 2000 g for 10 min. Plasma was collected into cryogenic vials  and stored at −20 °C in Sierra Leone before transporting on dry ice (−80 °C) to the Galicia Sur Health Research Institute in Vigo, Spain, for molecular analysis. HIV reverse transcriptase (RT), protease (PR), and integrase (IN) were amplified from virus-derived plasma using an in-house Sanger sequencing protocol as previously described [http://hivdb.stanford.edu, accessed on 21 June 2020). For each patient, 3 to 5 mL of venous blood were collected into tubes (BD Vacutainerescribed . FASTA sThe genotypic susceptibility score (GSS), which estimates the number of HIV active drugs in a given combination ART regimen, was calculated as follows: (1) susceptible and potential low-level resistance were scored as 1, (2) low-level and intermediate resistance were pooled as intermediate and scored as 0.5, and high-level resistance was scored as 0. The GSS was assigned by summing the individual resistance scores for each first-line drug in the combination ART regimen as we previously described . p < 0.05 was considered statistically significant.Statistical analysis was performed using the Statistical Package for the Social Sciences software . Categorical variables were presented as frequency (percentages) and were compared using Pearson’s chi-squared test. Continuous variables were expressed as median  and compared using the non-parametric Mann–Whitney U-test or Kruskal–Wallis test, where appropriate. 3 and 544 (IQR 260–849) cells/mm3, respectively, with 100% of children and 93.6% of adolescents having acquired HIV through MTCT. All children and adolescents (100%) were ART-experienced, with a median treatment duration of 36 (IQR 12–60) months and 84 (IQR 60–108) months, respectively. The majority of children  and adolescents  were on first-line ART.A total of 197 PLWH were enrolled in the study, consisting of 96 children, 47 adolescents, and 54 pregnant women. For the pregnant women, the median age were 26 years (IQR 23–31) and the median CD4 count was 526 cells/mm3 (IQR 459–698). The majority  were ART-experienced with median ART duration of 3 months (range 1–96) and were receiving tenofovir/lamivudine/efavirenz, TDF/3TC/EFV . Over a quarter of the pregnant women  were new HIV diagnosis, with the majority of them  diagnosed during the second or third trimester of pregnancy. Plasma samples were analyzed from all 197 patients, and the HIV pol region was successfully sequenced in 108 patients, distributed as follows: 43 children, 33 adolescents, and 32 pregnant women. This yielded an overall sequencing success rate of 54.8% (108/197). The sequencing success rates by demographic groups were as follows: children, 44.8% (43/96); adolescents, 70.2% (33/47); and pregnant women, 59.3% (32/54).The majority of patients were infected with the HIV-1 subtype CRF02_AG , followed by subtypes G , A , and C .Genotypic resistance testing was performed on all 96 children (100%) and 47 adolescents (100%), which yielded 64 RT, 57 PR, and 67 IN sequences.Nearly 80% (51/64) of RT sequences from children and adolescents harbored RAMs to NRTIs and NNRTIs. n = 21/64), or in combinations of two , three , or five RAMs . On the other hand, 133 NNRTI RAMs were identified, either as a single RAM , or in combinations of two , or three  RAMs.A total of 117 NRTI RAMs were identified from these two groups and were distributed as follows: as single RAMs , K103N , Y181C/V/I , T215F/Y , V108I , A98G , G190A , K101E/H , M41L , L74I/V , and H221Y   a,b. n = 2), M46I/M (n = 1), and I84I/V (n = 1). The combination mutation I54V+V82A confers intermediate resistance to all the currently approved PIs with the exception of darunavir, which retains full activity. Interestingly, there was no documented record of prior exposure to PI-based ART in these 6 patients.We observed 4 PI-associated RAMs in 6 children/adolescents, as follows: the combination mutation I54V + V82A (n = 2), E157Q (n = 5), and S230SR (n = 1).Polymorphic accessory INSTI-selected mutations, which have minimal effect on INSTI susceptibility were observed in 8 children/adolescents, as follows: T97A ; and (2) 7 RT, 6 PR, and 9 IN from the newly diagnosed ART-naïve patients (n = 15). Genotypic resistance testing was performed on all 54 pregnant women (100%). A total of 34 RT, 27 PR, and 33 IN sequences were obtained, which were distributed as follows based on treatment status: (1) 27 RT, 21 PR, and 24 IN from ART-experienced patients (n = 26/34) of RT sequences harbored RAMs to NRTIs or NNRIs in pregnant women, which were distributed as follows: (1) ART-experienced , and (2) newly diagnosed ART-naïve . Overall, 76.5% (n = 1/39) or as a combination of two mutations ; and (2) 2 RAMs in the newly diagnosed ART-naïve, which occurred as a combination of two mutations . On the other hand, 18 NNRTI RAMs were identified, as follows: (1) 5 RAMs in the newly diagnosed ART-naïve patients, which occurred as a combination of two  or three mutations ; and (2) 13 RAMs in the ART-experienced patients, which occurred as a single mutation  or in combinations of two , three  or four mutations . A total of 9 NRTI RAMs were identified, as follows: (1) 7 RAMs in the ART-experienced group, which occurred as a single mutation , M184V , Y181C/V/I , P225H , K219N/E/Q/R , and K238T   a,b. n = 1), E157Q (n = 2), and G163R (n = 1), which have minimal effect on INSTI susceptibility, were observed in four pregnant women.No PIs RAMs were observed, whereas three accessory INSTI-selected mutations T97A  and NNRTIs (nearly 50%) in the three demographic groups, whereas the prevalence of RAMs to PIs and INSTIs was relatively low or absent. Our findings confirm the situation previously described in the WHO 2019 HIVDR report ,16,17,18Current international ART treatment guidelines recommend a dual NRTI-backbone in combination with an INSTI or a boosted-PI with a high genetic barrier to developing resistance such as darunavir/ritonavir (DRV/r) in the treatment of HIV-infected individuals ,19,20,21Despite its high potency, good tolerability, and high genetic barrier to developing drug resistance, safety concerns persist around the peri-conception use of DTG in women of child-bearing age and during the first trimester of pregnancy, due to the reported higher incidence of neural tube defects ,26,27. AWe found that M184V was the most frequent NRTI RAM, occurring with a prevalence of nearly 77% among children/adolescents and 11% of pregnant women. This mutation is selected by 3TC and emtricitabine (FTC), which significantly reduces their activity  while conferring low-level resistance to ABC ,37,38. IThe marked disparity in frequency of M184V-selected viruses among children and adolescents (77%) and pregnant women (11%) was noteworthy. Studies have shown that M184V is associated with reduced replicative activity in the absence of drug pressure, compared with the wild-type (WT) virus ,43,44. TThe prevalence of the NNRTI mutation K103N, which is selected by EFV and NVP was high in all three study groups. This finding is in line with reports from the SSA, where the frequency of K103 has exceeded 10% in most LMICs reporting HIVDR surveillance data to the WHO . There aOur study had several limitations. Firstly, our study enrolled a small sample size from a single clinical site, which limits the generalizability of study findings. Secondly, although the issues of pretreatment (PDR) and/or transmitted drug HIV drug resistance (TDR) remain important barriers to reducing vertical HIV transmission, we were unable to assess this fully due to the relatively small number of ART-naïve pregnant study population. Thirdly, due to insufficient plasma samples, VL measurement could not be performed for all patients. Finally, because the majority of the patients were on ART for a long period of time , we suspect that a substantial proportion of these patients were either virologically suppressed or had low viremia, which may have contributed to the low rate of sequencing success, especially among children. Nonetheless, our findings contribute to our current understanding of circulating HIV strains and HIVDR among three vulnerable populations that are disproportionately affected by the growing HIV epidemic and may have some policy implications for PMTCT program activities. Larger studies are needed to assess the countrywide HIVDR prevalence rates and associated factors to help inform treatment strategies and public health approaches in the national AIDS response in Sierra Leone.We observed a high prevalence of circulating RAMs to NRTIs  and NNRTIs (EFV and NVP) among HIV-infected children, adolescents, and pregnant women in Freetown, Sierra Leone. In contrast, the prevalence of RAMs to PIs and INSTI was low or absent. Although the three populations were not biologically related, our findings may have implications for the success of PMTCT efforts in the country. Furthermore, our findings suggest the need for a national HIVDR surveillance program to help guide ART choice and improve treatment outcomes for patients.