Insecticides and pesticides were also secondary sources of chemical poisoning. Additional research revealed that the incidence of food, chemical, and drug poisoning was highest in children aged 1–15 years ; patients aged 11–20 years had the highest incidence of chemical poisoning . Most poisoning incidents among youngsters are caused by easy access to drugs at home. Implementing strategies to enhance public awareness and limit children’s access to drugs would contribute considerably to decreasing the community’s burden of this problem. The findings of this study suggest that Al-Baha should improve its education regarding the rational and safe use of drugs and chemicals.Poisoning is a common and severe problem worldwide. Due to significant growth in the agricultural, chemical, and pharmaceutical industries over the past few decades, poisoning risks have increased with the use of food, chemicals, and medicines everywhere in the world, especially in Saudi Arabia. Advanced information on acute poisoning patterns is critical for the effective management of poisoning events. This study aimed to examine the characteristics of patients with various patterns of acute poisoning, caused by food, drugs, and chemicals, that were reported to the Department of Toxicology and Poison Center at King Fahad Hospital and the Poison Center in Al-Baha Province, Saudi Arabia. The study also examined the relationship between demographic characteristics, including age, toxin type, and geographical distribution, and poisonings in Baha Province. This retrospective cross-sectional analysis included 622 poisoning cases. The data were collected from 2019 to 2022 and it was found that out of 622 instances, 159 had food poisoning, with more men than females sick , 377 had drug poisoning , and 86 had chemical poisoning . This study found that the most prevalent agents implicated in acute poisoning were medicines, particularly analgesics and antipsychotic drugs. Food poisoning was the second most common acute poisoning, affecting largely males followed by female patients. Finally, chemical poisoning involved acute poisoning, with most cases involving methanol and household items including the strongest bleaches (chlorines) (Clorox Poisoning is a worldwide public health concern that is regarded as one of the most serious medical contingencies because its multitude varies from one location to another . PoisoniSalmonella and E. coli have been reported to be among the most important pathogens that have recently become more prevalent causing poisoning, particularly in children [Salmonella contamination being the second most common clinical manifestation [Acute contamination occurs when food is contaminated by microorganisms that aggravate toxicosis. children . Foodborestation . Followiestation . In deveestation . Becauseestation . The infestation ,11. Howeestation . Chemicaestation . Chemicaestation ,14.Overall, the effect of toxicity cases, whether from food contamination, prolonged drug administration, or chemical mishandling, varies with various factors such as age, nature of immunity, poison reception, toxicity estimation, and administration. In the past couple of decades, Saudi Arabia has achieved major milestones in agriculture, industrial technology, and medical pharmacology. These advancements have resulted in the development and widespread availability of a wide range of toxic agents, including pesticides, therapeutic drugs, and other chemicals, as well as significant changes in the trends of acute poisoning . Toxic aThis descriptive cross-sectional study and retrospective case-series study were performed to assess the various poisoning cases, particularly food, drug, and chemical poisonings. Data were collected from tertiary hospitals and poison centers located in Al-Baha Province in the Kingdom of Saudi Arabia. From 2019 to 2022, 622 poisoning cases were collected during the study period. We identified 159 patients with food poisoning, 377 patients with drug toxicities, and the remaining 86 patients with chemical poisoning out of the total number of poisoning cases. This study enrolled records of patients who complained of mild, average and serious symptoms of drug, chemical, and food poisoning. According to Persson’s poison severity score, individuals were classified as having mild, moderate and severe/serious cases of poisoning .Patient histories, physical examinations, and routine toxicological laboratory tests were used to make the diagnoses. An electronic hospital information system was used to obtain the relevant medical records. In a clinic, an electronic system is used to record patient information. Searching for some of the following keywords yielded various food, chemical, and drug poisoning cases: food poisoning, chemical and home-based chemical poisoning, and drugs/medication adverse effects. A structured form was used to collect and document demographic data such as age, sex, place of residence, diagnosis, type of exposure , type of toxic agent , and treatment outcome (whether the patient survived or died). Therapeutic drugs, pesticides, alcohol, poisonous fumes/gases/vapors, chemicals, food, other substances, and unknown compounds were classified as toxic agents. Symptomatic treatment was administered in the absence of specific antidotes. As a result, the toxic agents linked to fatalities and morbidity, as well as the pattern of acute poisoning, which varies from place to place and over time, are expected to change. There is a constant need to obtain updated information on acute poisoning to plan rational resource allocation and evaluate public health interventions. Epidemiological studies are urgently needed in each country and region to clarify the profile of acute poisoning and assist authorities in developing a plan for its prevention and control. Numerous studies have been conducted on acute poisoning in various Saudi populations. To our knowledge, no study on poisoning cases, whether food, chemical, or drug, has been conducted in Al-Baha City from 2019 to 2022.p < 0.05.The data were analyzed using the SPSS/PC statistical program. Simple frequency tables, as well as means and standard deviations, were used to generate descriptive statistics. Chi-square tests were used to compare categorical variables in inferential statistics. The statistical significance level was set at There was no involvement of patients or the general public.A total of 159 cases of food poisoning were reported to the toxicology center of King Fahad Hospital and the poison center regions of Al-Baha from 2019 to 2022. Based on the report of food poisoning, the highest percentage of poisoning was observed in males (85 cases) whereas 74 cases were female patients. The lowest age of the patient was found to be a one-year-old kid and highest age of the patient that was suffering from food poisoning was 89 years old. The patients most vulnerable to poisoning were those aged 1–10 years (105 cases) followed by patients aged 11–20 (17 cases) and above 40 years (16 cases) .Salmonella was the most common causative agent of infection (149 cases), followed by six cases that contained the Enterococcus species and four cases that contained E. coli. Salmonella infection can sometimes be caused by a variety of meat products, such as chicken, beef and eggs, fruits, sprouts, various vegetables, and even manufactured foods such as nut butters, chicken nuggets, and stuffed chicken meals [Enterococcus poisoning is less common among patients, hygienic conditions in the Kingdom are not jeopardized. Furthermore, E. coli infections are less prevalent in Al-Baha, implying that people do not consume more contaminated raw food, particularly raw meat and vegetables. Based on the geographic distribution of the cities in the Baha province, the city of Al-Baha comes in with the highest rates of food poisoning, with 109 subjects (68.5%) followed by 24 (15.1%) in Al-Agig. The incidence rate of food poisoning in Almandag, Algara, Bhaljureshi and Qilwa are ten (6.3%), six (3.8%) and four cases (2.5%), respectively. However, 136 (85.5%) poisonings associated with foodborne diseases were reported at home, whereas 23 (14.4%) cases were reported from the restaurants and weddings. Depending on the severity of the infection as determined by signs and symptoms and grading score, 67 patients were diagnosed with mild infection (42.1%) followed by 63 patients (39.6%) with an average infection. Serious infection was observed in 29 (18.3%) patients. The treatment regimens used in our study included antibiotics and supportive care.Furthermore, data analysis revealed that en meals . BecauseInnumerable antibiotics were administered according to the age groups. We observed that 78 patients were administered with Augmentin antibiotics to treat foodborne infections, with a percentage of 49.15%. Quinolones were then administered to 35 patients, with a success rate of 22.0%. Cephalosporins were administered as a therapeutic regimen in 29 patients (18.2%). Penicillin antibiotics were recommended to 17 (10.7%) patients. Depending on the signs and symptoms patients encountered during food poisoning, health care practitioners have discovered various diagnostic tools. Approximately 150 patients (94.3%) complained of common signs and symptoms such as diarrhea, fever, and stomach cramps. Six patients (3.8%) had severe signs and symptoms such as fever with chills, severe headache, severe abdominal pain, micturition, and nausea. Three patients (1.9%) reported diarrhea, stomach cramps, and occasional fever. According to a report on food poisoning from 2019 to 2022, the highest poisoning levels were documented in 2021, 68 (42.8%), followed by between 2019 and 2022, 34 (21.4%), and 2020, 23 (14.5%) .χ2 = 44.345 and p < 0.05) was observed between the various age groups of patients with respect to the type of organism causing food poisoning. Various bacterial infections were reported among patients of different ages starting from one year to 89 years old. It was noticed that around 104 cases of Salmonella were found in patients of an age group of between 1 and 10 years. Between patient ages of 11- and 20-years age it was noticed that only 17 patients had food poisoning because of salmonella contamination. Twenty-two Patients aged 31–89 years were infected with Salmonella, and six patients aged 21–30 years were infected with Salmonella. Enterococcus infection was rarely seen among patients suffering from food poisoning, as only two patients within the age of 21 to 40 were infected, and four patients aged ≥40 y had Enterococcus. This means that the infection was observed to be highest in children aged between 1 and 10 years. However, they may not be resistant to infection. Necessary precautionary measures should be taken to ensure the safety of children in this age group. A statistically significant difference  was observed in the severity of infections at different stages with respect to patient age. Patients aged between 1 and 10 years exhibited all stages of infection compared with other age groups . Forty-three patients aged 1–10 years, nine patients aged 11–20 years, four patients aged 21–30 years, two patients aged 31–40 years and five patients aged > 40 years had an average infection rate. Similarly, 41 patients aged 1–10 years, 8 patients aged 11–20 years, 5 patients aged 21–30 years, 7 patients aged 31–40 years and 6 patients aged > 40 years had mild infections. A total of 20 patients aged 1–10 years, 3 patients aged 11–20 years, 0 patients aged 21–30 years, 3 patients aged 31–40 years and 3 patients aged > 40 years had serious infections  was observed in the administration of antibiotics to various patients in different age groups. Augmentin is a mild antibiotic that was prescribed to 73 patients aged till 1 –10 years. Only five patients within the age group of 11–20 years were recommended Augmentin antibiotics. For older age groups (21–89 years), Augmentin was not recommended to the patients. A total of 73 patients were prescribed Augmentin, 21 patients were prescribed cephalosporins, 4 patients were prescribed quinolones and 7 patients were prescribed Penicillin within the age group 1 to 11 years of age. Similarly, five patients received Augmentin, five received cephalosporins, four received quinolones, and three received Penicillin from 11 to 20 years of age. Similarly, six patients received quinolones, and three received Penicillin from 21 to 30 years of age. Additionally, eleven patients received quinolones and one received Penicillin in the 31 to 40 years age group; three received cephalosporins, ten received quinolones, and three received Penicillin in the >40 years age group (χ2 = 103.678 and p < 0.001) was observed. Most cases had symptoms of diarrhea, fever, and stomach cramps in children aged one to eleven years, and severe signs and symptoms, such as fever with chills, severe headache, severe abdominal pain, micturition, and nausea, was observed only in a few patients , followed by patients aged 11–20 years . In the age group of 21–30 years, 74 patients (19.6%) were vulnerable to various kinds of drug toxicities. A total of 47 cases of drug toxicity were found in the age group of 31–40 years old with a 12.5% vulnerability rate, and 35 patients (9.3%) were above 40 years of age. In the present study, the incidence rate of drug poisoning in males was 204 (54.1%), while in females it was 173 (45.9%), and the male to female ratio was 1.2:1. The patients most vulnerable to poisoning were those aged 1–10 years  was the most common (92.83%), followed by inhalation  (5.30%) and other unknown routes (1.87%). Various drugs caused different adverse effects when administered depending on the severity of the infection. Furthermore, we found that out of 377 patients with drug toxicity, 376 recovered and one died. Of these, 360 were Saudi and 17 were non-Saudi. In 2019, 146 patients (38.7%) suffered from drug poisoning symptoms; in 2020, 76 (20.2%) had drug poisoning; in 2021, 122 patients (32.4%) had drug poisoning; and in 2022, 33 patients 33 (8.8%) had adverse drug effectsThe most prevalent drugs and other therapeutic drugs that cause severe adverse events are shown in χ2 77.301 and p value < 0.001. Patients of different age groups were investigated to determine impact of various drugs. A statistically significant difference was observed between different age groups and drug toxicity. Drug poisoning was common in both men and women aged 10–30 years. However, drug poisoning was more common in men, with a total number of 204 cases, compared to women, which were 173 total cases in number. There was a statistically significant difference noticed between gender and the type of the drug they consumed, with a chi square value χ2 of 215.236 was observed, and a significant difference (p < 0.05) was found between the different age groups. It was noted that the administration of N-acetyl cysteine and activated charcoal were administered in 39.33% (n = 83) and 30.06% (n = 65) of cases, respectively, to prevent the absorption of toxic agents. In addition, 6.6% of the patients received naloxone as an antidote, 5.76% received omeprazole, 6.16% received sodium bicarbonate, 4.735 received glutamate  were recently reported to cause adverse effects in patients aged 21–30 years old. Precautionary measures should be taken to stop the trend of severe adverse effects when using recreational drugs. A statistically significant difference was found, with patients showing notable variations in their age with respect to the drugs administered. A chi value lutamate .Most patients complained of severe signs and symptoms such as abdominal movement, dystonia, sleeping disorders and epigastric pain. These symptoms were followed by other serious symptoms, such as headache, nausea, vomiting, dizziness, confusion spasm and disorientation. Other symptoms that were observed at lower frequencies in patients were watery diarrhea and seizures.n = 41) of all chemical poisoning cases involved children in the group of 11–20 years old. This group was followed by 1–10-year-old children, whose contribution was 42% (n = 36) (n = 1). Methanol toxicity along with unknown drug administration was the most common chemical poisoning agent observed, with a frequency of 21 patients constituting 24.4% of the total 100%. Among the various cleaning agents, Clorox was the second most common cause of chemical poisoning in 16 patients (18.7%). Spraying of insecticides and pesticides also caused severe chemical poisoning, which was observed in 15 patients constituting 17.2% of the aggregate. In addition, alcohol poisoning (10.5%), hair dye poisoning (3.5%), and other chemicals were also observed  were in males and 22 (25.6%) were in females, and the male to female ratio was 3:1. Almost 48% ((n = 36) . In the observed . In the Among the various routes of exposure, swallowing was the most common (68.6%) route followed by inhalation  (24.4%). The dermal and injection routes constituted approximately 4.75 and 2.3%, respectively. Various drugs caused different adverse effects when administered depending on the severity of the infection. In our study, we noticed that among 76 patients, 42 (48.8%) had average infection, 28 (32.5%) had serious infection, and 16 (18.65%) had mild infection. Of the 86 patients with chemical toxicity, 85 recovered and one died. Of these, 75 were Saudi and 11 were non-Saudi. In 2020, 37 (43%) patients had chemical poisoning; in 2022, 19 patients (22.1%) had chemical poisoning; in 2022, 15 patients (17.4%) had chemical adverse effects; and in 2021, 15 patients (17.4%) suffered from chemical poisoning symptoms.χ2 = 8.294 and p value < 0.05. A significant value was observed in the outcome to exposure with respect to gender . We found that 63 male cases out of 86 were accidental and only one was suicidal, compared to 18 female cases of accidental chemical exposure and four suicidal. Various therapeutic regimens were administered in the form of antidotes, as shown in χ2 = 45.94 and a p value < 0.001A statistically significant difference was observed between sex and chemical toxicant exposure, meaning that males were more exposed to toxic chemicals than females. In addition, we also correlated the ROA with males and females and found that males were exposed to chemical contaminants more through swallowing than females at ratio of 5:1, followed by inhalation (1:1), dermal (2:1) and injection (1:1). The chi- square value was p < 0.01 with a χ2 value of 82.171 was found in patients when the type of toxicant was corelated with various age groups. Moreover, only five suicidal cases were found between 1 and 20 years of age. Almost all cases of chemical poisoning were accidental. A statistically significant difference was also observed in the route of administration and treatment regimen when their effect was observed in various age groups  was the most prevalent home product implicated in chemical poisoning. Among the recorded instances, activated charcoal was the most effective treatment method. It is well recognized that administering activated charcoal to patients who have recently overdosed on organochlorine pesticides and herbicides considerably limits their total medication absorption. Additionally, it can be used to reduce the absorption of toxic and narcotic overdoses through gastrointestinal decontamination. The oral route is the most prevalent type of exposure reported in the present study, and it is conceivable that this was because patients between the ages of 11 and 20 years old were those who reported the most occurrences of unintentional medication ingestion. The results of this study also showed a strong correlation between the ages of those in the study and the types of agents used in poisoning cases. However, despite some of the prior literature supporting this conclusion, several other studies contradict it. Additionally, because no consistent pattern can be observed, it is challenging to pinpoint the cause of these changes using the number of instances for each type. Furthermore, our investigation showed that the most common toxicants used were prescription medications. In both industrialized and developing nations, medicinal medications have been shown to follow a similar trend as the most frequent cause of acute poisoning. Sedatives/hypnotics are often consumed in Taiwan, Hong Kong, Iran, and Finland with regards to the subgroup of therapeutic medications in poisoning cases, whereas analgesics are most frequently consumed in Turkey and the USA [Additionally, unintentional chemical poisoning has been reported in several studies . It is i the USA . The maj the USA . However the USA . AdditioThese findings add to our understanding of the prevalence and incidence of acute poisoning in Al-Baha. The results revealed that more men than women experienced acute poisoning, with young adults aged 1–20 years being the most vulnerable. We also discovered that the vast majority of poisonings were unintentional, with very few intentional poisonings. The results of the current study showed that pharmaceutical and chemical items were the primary causes of poisoning in Al-Baha. According to our findings, therapeutic drugs were the most commonly implicated group of toxic agents, followed by pesticides and alcohols, particularly methanol. Poisoning from analgesics, anticonvulsants, and antipsychotics was more common than poisoning from other therapeutic drugs. Furthermore, the incidences of disinfectant poisoning have significantly increased. Based on the findings of the current study, the following recommendations are made. Firstly, vulnerable groups, such as young adults, require special attention due to their limited coping abilities. To reduce acute poisoning, more investment is needed to promote public health education on the rational use and safe storage of toxic agents, as well as self-protection. Patients who attempt suicide should seek psychiatric help as soon as possible. In suicide patients, early psychiatric consultation and identification may reduce the risk of further self-harm. In general, relevant policies and regulations should be developed and implemented as soon as possible to limit access to toxic agents, particularly pesticides, owing to their high toxicity. Furthermore, to prevent the high incidences of poisoning among children and adolescents, parental awareness should be increased, and educational programs should be made available to the general public.


Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox reactions, as they are essential components of the engine that life uses to tap into the thermodynamic disequilibria necessary for metabolism. The number of proteins that evolved to catalyze redox reactions is extraordinary, as is the diversification level of metal cofactors and catalytic domain structures involved. Notwithstanding the importance of the topic, the relationship between metals and the redox reactions they are involved in has been poorly explored. This work reviews the structure and function of different prokaryotic organometallic–protein complexes, highlighting their pivotal role in controlling biogeochemistry. We focus on a specific subset of metal-containing oxidoreductases (EC1 or EC7.1), which are directly involved in biogeochemical cycles, i.e., at least one substrate or product is a small inorganic molecule that is or can be exchanged with the environment. Based on these inclusion criteria, we select and report 59 metalloenzymes, describing the organometallic structure of their active sites, the redox reactions in which they are involved, and their biogeochemical roles. To precisely and efficiently transfer electrons to and from a wide range of molecules, they finely tune their conjugated metals' midpoint electric potential by controlling the coordination sphere, geometry, and accessibility of the active site . Element., carbon, hydrogen, nitrogen, oxygen, and sulfur, also known as CHNOS elements) is primarily carried out by microorganisms inhabiting diverse ecosystems , codh [1MGJ], 3S4] cluster rather than being bridged to a cubane [Fe4S4] [The carbon monoxide dehydrogenase ( [Fe4S4] .pmoB1/B2 [3RGB]), which uses Cu as a catalytic cofactor, and the cytoplasmic, copper starvation-induced soluble Methane monooxygenase  gas and it is present in significant quantities also in the mantle. This element is vital in building nucleic acids, proteins, and enzymes. At the enzymatic level, nitrogen can be transformed between different compounds with different redox states, e.g., NH4+, NO, NO2, N2O, NO3−, NO2−, hydroxylamine, and amino acids, moving from +5 in NO3− to -3 in ammonia 36–38]. Tnditions , showingve force ,45. Thise.g., Fe/Mn, Cu, and Ni)  [On the other hand, aerobic respiration induces the formation of reactive species of oxygen (ROS) B, which gure S2) ,50. Hydrgure S2) .2 regeneration through the reverse reaction [Hydrogen is a key reduced compound in the redox balance of the planet. It is produced by several abiotic processes, including water photolysis/radiolysis, hydrothermal reactions, magmatic degassing, and hydration of iron-rich ultramafic rocks . Hydrogereaction ,55  [., Mn2+/Mg2+/Fe2+ and Mo/W). In this context, excluding the wrong metals from proteins may be more challenging than acquiring the right ones [2+, [Diverse factors constrain the choice of metals at the core of metabolisms: the environmental availability of the element of interest, its suitability for the specific redox reaction to be catalyzed, and the ability to control its binding to the target enzyme. Theoretically, metal-binding affinities of natural proteins are defined by the ligand field stabilization energy of metal ions and follow the Irving–Williams (IW) series (Mn > Zn2+) . In prac > Zn2+)  so that  > Zn2+) . The envght ones , and havnes [2+,  could ree.g., Fe-S proteins, [At the environmental level, the (bio)availability of metals might control to a first order its utilization by biology . Howeverroteins,  relying in vivo utilization of metal ions by biomolecules is challenging since complicated metal centers can remain poorly defined even after structure determination due to, e.g., experimental procedure-related substitutions.Currently, biogeochemical cycles are dominated by Fe as a key catalytic metal  reactions are lacking, and a number of theoretically possible reactions have yet to be identified in nature [Life sustains itself through redox reactions that capitalize on environmental thermodynamic disequilibria. A reasonable hypothesis is that life became proficient in redox reactions as it evolved. In prebiotic Earth, metal ions alone were sufficient redox agents. As the concentration of organic molecules in the environment increased, organometallic complexes formed. The existence of these complexes, in turn, created the context for metallopeptides evolutions that eventually developed into metalloproteins . Extant n nature .The requirement of life for metals as cofactors in key biogeochemical reactions attests to the vital role that metals play in the functioning of Earth and the intricate relationship between the biosphere and the geosphere. Complex stellar processes, protoplanetary disk accretion, and planetary differentiation , changinThere is a universal need for redox chemistry by life to use thermodynamic disequilibrium.Biogeochemical cycles, and therefore the functioning of our planet, are controlled by a small number of biogeochemically relevant redox proteins, most of which use metal cofactors. The metal used is generally tuned together with the protein structure to the midpoint potential of the reaction catalyzed, although alternatives are possible.Metal choice is dictated on the first order by availability and active transport and refined by protein structure. Besides, evolution contributed to ‘frozen accidents’ that irreversibly paired some metals to specific cycles.The correlation between the diversity of metal cofactors and the biogeochemical redox reactions in which they are involved is still unclarified.Despite their importance for our planet’s functioning, we have limited information regarding the organometallic structure found in oxidoreductases of uncultured lineages of microorganisms.Click here for additional data file.


Background: The maturation of cardiomyocytes is a rapidly evolving area of research within the field of cardiovascular medicine. Understanding the molecular mechanisms underlying cardiomyocyte maturation is essential to advancing our knowledge of the underlying causes of cardiovascular disease. Impaired maturation can lead to the development of cardiomyopathy, particularly dilated cardiomyopathy (DCM). Recent studies have confirmed the involvement of the ACTN2 and RYR2 genes in the maturation process, facilitating the functional maturation of the sarcomere and calcium handling. Defective sarcomere and electrophysiological maturation have been linked to severe forms of cardiomyopathy. This report presents a rare case of DCM with myocardial non-compaction, probably resulting from allelic collapse of both the ACTN2 and RYR2 genes. Case Presentation: The proband in this case was a four-year-old male child who presented with a recurrent and aggressive reduction in activity tolerance, decreased ingestion volume, and profuse sweating. Electrocardiography revealed significant ST-T segment depression . Echocardiography showed an enlarged left ventricle and marked myocardial non-compaction. Cardiac magnetic resonance imaging revealed increased left ventricular trabeculae, an enlarged left ventricle, and a reduced ejection fraction. Whole exome sequencing revealed a restricted genomic depletion in the 1q43 region , encompassing the coding genes ACTN2, MTR, and RYR2. The identified variant resulted in heterozygous variations in these three genes, with the ACTN2 g.236,686,454-236,764,631_del and RYR2 g.237,402,134-237,833,988_del variants being the dominant contributors to the induction of cardiomyopathy. The patient was finally diagnosed with DCM and left ventricular myocardial non-compaction. Conclusions: This study reports a rare case of DCM with myocardial non-compaction caused by the allelic collapse of the ACTN2 and RYR2 genes. This case provides the first human validation of the critical role of cardiomyocyte maturation in maintaining cardiac function and stability and confirms the key findings of previous experimental research conducted by our group. This report emphasizes the connection between genes involved in regulating the maturation of cardiomyocytes and the development of cardiomyopathy. TTN TTN [DES ES [LMNA  and RBM2A [RBM20 . MoreoveA [RBM20 ,7,8. We ACTN2, MTR, and RYR2. This variant resulted in heterozygous variants in these three crucial genes. The variants ACTN2 g.236,686,454-236,764,631_del and RYR2 g.237,402,134-237,833,988_del were found to contribute to cardiomyopathy in a dominant manner, while MTR is usually associated with neurodevelopmental disorders in a recessive manner. This is a rare case of heterozygous loss-of-function variants in both ACTN2 and RYR2 genes, offering an opportunity to study the clinical manifestation and phenotype when sarcomere and calcium handling maturation are impaired in cardiomyocytes. This rare case also demonstrated DCM with left ventricular myocardial non-compaction which may be due to ACTN2 and RYR2 variants, validating previous experimental findings. However, it is a pity that there is no way to build a similar mouse model to test the variants.In this report, we present a novel case of restricted genomic depletion in the 1q43 region , which includes the coding genes This study had been approved by the Ethics Committee of the West China Second Hospital of Sichuan University . Informed consent was obtained from the patient’s parents before performing whole exon sequencing and for the inclusion of the patient’s clinical and imaging details in subsequent publications.This patient was a 4-year-old boy who was admitted to our hospital due to a decrease in activity tolerance for 8 days, as well as decreased ingestion volume and heavy sweating. The child also reported shortness of breath, persistent cough, palpitations, and a pale complexion. However, his parents reported no symptoms of cyanosis, fever, seizures, edema, vomiting, diarrhea, oliguria, or abdominal pain for this patient. The patient did not have any history of chest pain or syncope. At birth, the proband was full-term with a gestational age of 38 weeks and a birth weight of 2900 g. Although there was some growth retardation observed during development, the child’s cognitive and motor abilities were normal. On examination, the patient appeared acutely ill with severe diaphoresis and fatigue. His heart rate was 115 beats per minute and had irregular premature beats. Blood pressure was elevated in the left upper limb, but normal in the other three extremities. The patient also showed signs of slight malnutrition but had a normal response to external stimuli.On physical examination, the patient had no trauma injuries and had symmetrical respiratory movements in both lungs. Breath sounds were rough, and a diffuse small wheezing was heard in both lungs. The apex of the heartbeat was located in the left lower quadrant and the heart boundary was slightly enlarged. The heart rhythm was heterogeneous and had occasional premature beats. Heart sounds were dull, and no murmurs were heard. The abdomen was soft and medium in texture, and the spleen was not palpable. Muscle strength and tension in all four extremities were normal, and there were no pathological signs or meningeal irritation.The patient’s past medical history was unremarkable and there was no recent history of viral infection. The patient’s parents reported no history of illnesses, especially in the cardiovascular system, and no family history of cardiac attacks or cardiovascular diseases. There was no history of hypertension or coronary artery disease in the family.The initial laboratory results showed normal results for routine blood cell tests, blood gas analysis, hepatic and renal function. Elevation of B-type natriuretic peptide  was observed, while the serum level of cTnI was within the normal range. Negative results were obtained for rheumatic screening, autoimmune antibodies, and thyroid function. Screening for potentially infectious viruses, including SARS-CoV-2, coxsackie virus, adenovirus, influenza virus, rhinovirus, and respiratory syncytial virus, all resulted in negative results. Electrocardiography (ECG) showed significant ST-T segment depression  was performed to identify any crucial genetic variants. The WES analysis revealed a large-scale depletion of bases at the 1q43 site in the proband. The genomic location of the variant was located at chr1: 236,686,454-237,833,988/Hg38, encompassing the ng genes A. The AC,988_del B,C. The MTR gene is inherited in a recessive manner. Most reported cases of MTR-associated disorders are due to homozygous or compound heterozygous loss-of-function variants, suggesting that haploinsufficiency of the MTR gene is not a mechanism of disease. On the other hand, ACTN2 and RYR2 have been associated with cardiovascular disease through dominant inheritance patterns. Hence, haploinsufficiency should be considered for these two genes if loss-of-function alleles are identified. In the present case, the heterozygous variants in ACTN2  and RYR2  could account for the clinical manifestation in the proband. We believe that this should be the genetic cause of the disease in this proband.According to the OMIM and ClinGen databases, the After further evaluation, the patient was diagnosed with DCM and LVNC. To manage the condition, captopril, metoprolol, and digoxin were administered daily. Echocardiography and ECG were conducted every three months, and yearly cardiac magnetic resonance imaging (CMR) was performed. The follow-up results showed a persistently dilated left ventricle with non-compaction myocardium, with a left ventricular ejection fraction (LVEF) of 41%. Ultimately, heart transplantation was deemed the best alternative for the patient. ACTN2 and RYR2 are genes that play crucial roles in maintaining the structure and function of cardiomyocytes. ACTN2 mutations have been identified in patients with DCM and LVNC. These mutations disrupt the interaction between ACTN2 and the Z-disk protein LIM domain myoglobin, leading to sarcomere dysfunction and regulation problems. This results in a loss of mechanical support and contractile ability in cardiomyocytes, contributing to the decline in their number and maturation, which manifests as LVNC and DCM. LVNC is a rare and severe hereditary cardiomyopathy characterized by prominent trabecular cavities and deep intrusions into the left ventricular cavity due to a failure in the embryonic myocardial compaction process [ACTN2 and RYR2 are both critical genes in cardiomyocytes [ACTN2 and RYR2 serve as a dominant effect. Moreover, based on our previous research, the homozygous deletion of ACTN2 or RYR2 would induce a fetal-lethal condition. Thus, such two genes are of great importance, and haploinsufficiency had been identified in associated patients and animal models [ACTN2 is considered a major contributor to LVNC. In addition, ACTN2 was mentioned as the causative gene in patients with myopathy in previous studies. Therefore, even though the proband was absent from any abnormality in muscle strength and muscle, we could not easily exclude any impairments of skeletal muscle system of the proband in this case, and scheduled follow-up was required. RYR2 is a protein expressed on the sarcoplasmic reticulum (SR) in cardiomyocytes and is critical for releasing calcium ions. The most commonly associated cardiovascular disease with RYR2 mutations is catecholaminergic polymorphic ventricular tachycardia (CPVT). These mutations lead to abnormal calcium handling in the endoplasmic reticulum, abnormal hyperphosphorylation of PKA and CaMKII binding sites, and increased sensitivity to adrenaline, which increases diastolic calcium levels and drives calcium exchange through the sodium–calcium exchanger (NCX1). This increased calcium exchange affects the stability of the cellular membrane potential and can trigger arrhythmia-related manifestations, such as CPVT. RYR2 mutations have also been identified in some patients with cardiomyopathy, though the mechanism behind this association is not well understood. Given its dominant role in cardiomyocytes, further investigation is needed to confirm the role of RYR2 in related cardiomyopathies. In this case, the proband’s level of RYR2 expression has reduced, but the rest part of expression still acts on the Ca2+ ion channel. Thus, we supposed that this also has a certain impact on cardiomyopathy. As mentioned above, we cannot currently construct a similar mouse model to validate this event. In animal model research, the single base variant of RYR2 would induce CPVT or deadly arrhythmias. However, large depletion of RYR2 leads to severe DCM and impaired myocardial function [ACTN2 variant were also related with DCM and LVNC [ACTN2 and RYR2. process . Affectemyocytes ,8. Accorl models ,10,11. Afunction ,13,14. Mand LVNC ,11,15. AACTN2 and RYR2 are two key molecules responsible for functional maturation of the sarcomere and calcium handling, respectively. In a study conducted by Guo et al., the regulation mechanism of ACTN2 and RYR2 was first revealed, and their specific role in cardiomyocyte maturation was documented [ACTN2 and RYR2 genes leads to severe cardiomyopathy. Loss of ACTN2 results in a decrease in MRTF and impaired SRF signaling, leading to a disorder of morphological maturation [RYR2 leads to ER stress and the translocation of ATF4 and ATF6 into the nucleus, which promotes the expression of DDIT3 and triggers autophagy-mediated protein degradation and cardiomyocyte atrophy [ACTN2 and RYR2 in cardiomyocytes. It is noteworthy that we encountered a human patient with loss-of-function mutations in both ACTN2 and RYR2, who exhibited severe and early onset DCM. This clinical case validates the findings from our mouse studies. In addition, LNVC was associated with variants in other genes, including TTN, DES and RBM20. Variants in TTN in LVNC had been reported with a highly heterogeneous prevalence, and it had been confirmed that there was a haploinsufficient disease mechanism in titin truncation mutation cardiomyocytes, and that mitochondrial function would be impaired [DES variants were identified in patients with an unexplained etiology of cardiomyopathy, and most of the DES variants were recorded in LNVC. Moreover, an assessment of the mitochondrial function in four probands heterozygous for a disease-causing DES variant confirmed a decreased metabolic capacity of mitochondrial respiratory chain complexes in myocardial muscle [RBM20 and LVNC beyond DCM [RBM20 would lead to both DCM and LVNC. Thus, the mechanisms of RBM20 in regulating microstructure of cardiomyocytes required further analyses [Based on the principles of cardiac development, maturation plays a crucial role in realizing the full biological function of cardiomyocytes. In recent years, our group and William T. Pu have identified several molecular mechanisms involved in regulating cardiomyocyte maturation ,7,8,16. impaired ,18. DES l muscle ,20. Furtyond DCM . Howeveranalyses .RYR2 gene. Although the proband in this case carries a duplication of exon 1 in RYR2, it is yet to be determined if it can also cause abnormalities in myocardial development and structure, leading to LVNC or LVNC-CPVT overlap syndrome. This highlights the importance of further follow-up. The findings suggest that loss-of-function variants of RYR2, beyond single base mutations, can induce cardiomyopathy. This case provides a unique opportunity to investigate a tightly restricted scale depletion between ACTN2 and RYR2 genes, which is restricted to myocardial disorders, and further our understanding of the maturation process in cardiovascular diseases.Previously, numerous reports have documented patients with 1q43 depletion. However, these patients displayed a large-scale sequence depletion in the region spanning from 1q42 to 1q44, which encompassed many coding genes. Most of these cases presented with neurological disorders as the primary manifestation. Studies by Thomas Rosto et al.  and SeikACTN2 and RYR2 genes. Cardiomyocyte maturation, which is regulated by ACTN2 and RYR2, is critical for maintaining cardiac function and stability. The patient, who was diagnosed with this rare condition at a young age, highlights the importance of cardiomyocyte maturation in the development of cardiomyopathies. This case report is the first of its kind to validate the findings of our previous experimental research, emphasizing the crucial role of ACTN2 and RYR2 in cardiomyocyte maturation and the onset of cardiomyopathies. The report also sheds light on the critical genes involved in the regulation of cardiomyocyte maturation and their relationship with cardiomyopathies.This study presents a unique case of DCM with myocardial non-compaction, caused by the simultaneous collapse of


Anxiety and depression are associated with the risk of illness, presence of physical symptoms, and poor health in the COVID-19 epidemic. Our aim is to assess the severity of anxiety and depression in rheumatoid arthritis (RA) patients treated with disease-modifying anti-rheumatic drugs during the COVID-19 pandemic.The study is a longitudinal, hospital-based survey study including 102 RA patients receiving disease-modifying anti-rheumatic drugs with a mean of 55,2±11,9 years. Demographic data, educational status, marital status, employment status, economic status, patients with psychiatric disorders (with the use of prescribed medication for treatment), and medications were recorded. The severity of depression and anxiety were evaluated with the Beck Anxiety and Depression Inventory at the first and second visit of the follow-up during the pandemic period.The mean Beck depression inventory score was found to be higher in the conventional synthetic DMARDs group than in biological DMARDs . 46  had mild to severe anxiety symptoms in RA patients treated with conventional synthetic DMARDs, on the first visit. There was no significant difference in anxiety and depression status between the first and second visits. The difference in anxiety and depression symptoms between RA patients receiving conventional synthetic and biological DMARDs does not attain statistical significance. Also, no significant differences were found in anxiety and depression scores in the comparisons for gender, education, marital, working, and economic status.The severity of depression and anxiety were higher in RA patients receiving conventional synthetic DMARDs and biological DMARDs during the COVID-19 pandemic. Also, RA patients are likely to experience anxiety and depression during the period of the pandemic. Clinical manifestations can range from asymptomatic disease to fever, fatigue, myalgia, dry cough, dyspnea, and respiratory failure.1 Pneumonia, acute respiratory distress syndrome, sepsis, cardiac complications, and secondary infections are among the important causes of morbidity and mortality in SARS-CoViD-2.2 Other factors affecting morbidity and mortality in viral infections were reported as mood disorders, anxiety, post-traumatic stress syndrome, panic disorder, and bipolar disease.3 Depression and psychological stress decrease cell-mediated immunity and increase inflammatory processes during infections.3In late December 2019, a novel coronavirus called SARS-CoViD-2 (Severe Acute Respiratory Syndrome Coronavirus-2) was identified series of pneumonia cases in Wuhan City, China. This virus has spread all over the world, causing a global epidemic.4 Patients with hepatitis C virus (HCV) have fatigue, depression, and cognitive dysfunction.4 In addition, depression, anxiety, sleep disorders, psychosis, and delirium have been associated with pegylated interferon alfa-2a treatment in patients with HCV.4 Depression, anxiety, delirium, dementia, and manic-spectrum disorders have also been reported in patients with human immunodeficiency virus depending on the disease itself or the agents used in its treatment.4 The SARS outbreak in 2003 led to general stress, post-traumatic stress syndrome, and depressive disorder directly or indirectly in infected patients.6 In the SARS epidemic, 35% of patients had “moderate to severe” or “severe” and/or depressive symptoms after 1 month of recovery.7 The disease itself, the uncertainty about the efficacy of treatment, negative news in the media, the quarantine environment, the concern of infecting their loved ones, and high death rates cause increased stress in patients.8 In addition, the economic impact also contributes to depressive symptoms and levels.5 The medical history for psychiatric illness, loss of family members in the epidemic, and the sense of stigma associated with the disease were thought to be associated with neuropsychiatric problems in the Middle East respiratory syndrome outbreak.9Viral diseases are included in the etiology of some neuropsychiatric disorders.10 Also, not having enough surgical masks and being uncomfortable working from home are associated with poor mental health.11 Post-traumatic stress syndrome, depression, and anxiety have been reported in COVID-19 patients even after hospitalisation. It has been thought that these conditions occur due to the immune response to the virus, social isolation, the psychological impact of a new severe and potentially mortal disease, the anxiety of contagion to others, and stigmatisation.12 Factors such as difficulties in daily life, self-confidence in coping with stress, and expected health status are associated with depression in rheumatoid arthritis (RA) patients.13 In another study, there has been reported a relationship between poor sleep quality and anxiety, depression, self-efficacy, and stigma in RA patients.14 Here, sleep disturbance was found to be positively correlated with age, pain, disease activity, depression, anxiety, and stigma, while self-efficacy was negatively correlated with sleep disturbance.14Anxiety and depression are associated with the risk of illness, presence of physical symptoms, and poor health in the COVID-19 epidemic.15 RA patients treated with immunosuppressive drugs during the pandemic are at risk for anxiety and depression.In a cross-sectional study, it has been determined that depression is associated with social determinants of health, such as food insecurity in RA patients.In this study, we aim to evaluate the changes in the severity of depression and anxiety in patients with rheumatoid arthritis treated with disease-modifying anti-rheumatic drugs during the COVID-19 pandemic.16 We excluded patients with any connective tissue disease other than RA, infections, and hematological and solid malignancies were excluded from the study. Age, gender, disease duration, disease activity score-28 (DAS-28), educational status, marital status, employment status, economic status, comorbidities, patients with psychiatric disorders (with the use of prescribed medication for treatment), and rheumatological medication  of the patients were recorded. The study protocol was approved by the Faculty of Medicine Ethics Committee and designed consistent with the Declaration of Helsinki .The study is a longitudinal, hospital-based survey study including 102 rheumatoid arthritis patients. All patients aged 18 years and older included in the study were diagnosed with RA according to the 2010 American College of Rheumatology classification criteria.17 The validity and reliability of the Beck Depression Inventory for the Turkish population were tested by Hisli.18 Beck Anxiety Inventory consists of 21 self-reported items rated from 0 to 3 to assess the anxiety during the past week. The total score varies between 0 and 63 and indicates different severity degrees of depression .19 Ulusoy et al. validated the Turkish version of the Beck Anxiety Inventory.20Beck Depression Inventory consists of 21 items to evaluate and measure the severity of depression, and each item indicates a behavioral situation specific to depression. Items consisting of 4 sentences graded from low to high (0–3 points) are evaluated as follows: 0–9 as no/minimal, 10–18 as mild, 19–29 as moderate, and 30–63 as severe depression. Statistical analyses were performed with Statistical Package for Social Sciences (SPSS) for Windows . The data were presented as mean±standard deviation, median (25–75 percentile), frequency (n), and percentages (%). Categorical variables were analyzed by using Pearson’s chi-square test and Fisher’s exact test. A Kolmogorov-Smirnov test was used to determine the homogeneity of the data. Continuous variables were analyzed by using the Mann-Whitney U test and Independent-samples T-test according to the distribution. The Wilcoxon signed-rank test was used to compare two related samples. Multivariable regression analysis was performed to correlate demographic data with anxiety and depression. Regression analysis was performed for follow-up anxiety and depression symptoms as dependent variables and demographic data, duration between the two assessments and baseline anxiety and depressive data as independent variables. P-values of less than 0.05 were considered statistically significant.Table 1.A total of 102 patients, including 24 male  and 78 female  were included in the study. The mean age of the patients was 55,2±11,9 years (19–75 years) and the mean DAS-28 was 4,0±0,9 in patients treating with conventional synthetic DMARDs and 3,8±0,9 for biological DMARDs group. The mean Beck Anxiety Inventory score of the patients who were treated with conventional synthetic DMARDs was 13,1±11,6. The mean score was higher than in RA patients treated with biological DMARDs. The mean Beck depression inventory score was higher in the conventional synthetic DMARDs group than biological DMARDs . However, no significant differences were found in anxiety and depression scores between the two groups. Also, there were no statistically significant differences in the Beck Anxiety Inventory and Beck Depression Inventory scores in the comparison of gender, education status, marital status, employment status, and economic status of all patients. Demographic characteristics, anxiety, and depression scores of the RA patients are shown in All RA patients completed the questionnaires at the first and second visits during the pandemic period. The mean follow-up between the first visit to the second visit was 12,6±3,9 weeks for conventional synthetic DMARDs and 10,4±3,7 weeks for biological DMARDs. According to the Beck Depression Inventory score, 46 (65.7%) had mild to severe anxiety symptoms and 42 (60%) had mild to severe depression symptoms in RA patients treating with conventional synthetic DMARDs, on the first visit. At the second visit, 45 (64.2%) of the patients had anxiety symptoms and 38 (54.2%) had depression symptoms. According to the Beck Depression Inventory score, 46  had mild to severe anxiety symptoms and 42 (60%) had mild to severe depression symptoms in RA patients treating with conventional synthetic DMARDs on the first visit.Table 2 and Table 3.At the second visit, 45 (64.2%) of the patients had anxiety while 38 (54.2%) had depression. The anxiety levels of the biological DMARDs group at the first visit were as mild, moderate, and severe in 10 , 2 , and 6  patients, respectively. At the second visit, the rates were 10 (35.7%), 1 (17.1%) and 7 (11.4%), respectively. The severe depression ratio was higher in RA patients receiving biological DMARDs compared to conventional synthetic DMARDs . There was no significant difference in anxiety and depression between the first and second visits. Also, no significant differences were found in depression and anxiety status between both conventional and biological agent groups. The distribution of the Beck Anxiety Inventory score and Beck Depression Inventory score are shown in In the conventional synthetic DMARDs group, the mean Beck Anxiety Inventory scores were 13,1±11,6 at the first visit and 12,5±11,2 at the second visit. The ratios were 12,6±11,4 and 12,7±11,9 for the biological DMARDs group, respectively. The mean Beck Anxiety Inventory score and Beck Depression Inventory score were found to be decreased at the second visit compared to the first visit for both the traditional synthetic and biological DMARDs group. However, no significant differences were found in scores between visits. Positive correlation was found between DAS-28 and Beck Anxiety Inventory score  and Beck Depression Inventory scores . In the multivariable regression, higher Beck Anxiety Inventory score and Beck Depression Inventory scores at the first visit, and DAS-28 were independent risk factors for anxiety and depression at follow-up in both treatment groups.In the study, we aimed to evaluate the changes in the severity of anxiety and depression in patients with RA treating with Disease-Modifying Anti-Rheumatic Drugs during the COVID-19 pandemic. The mean Beck Anxiety Inventory score was higher than those who were treated with biological DMARDs compared to conventional synthetic DMARDs. The mean Beck Depression Inventory score was higher in the conventional DMARDs group than biological DMARDs. The mean Beck Anxiety Inventory score and Beck Depression Inventory score were found to be decreased at the second visit compared to the first visit for both DMARDs group. There was no statistically significant difference in Beck Anxiety Inventory score and Beck Depression Inventory score for gender, marital, employment, economic and educational status in all RA patients.5 The prevelance of depressive symptoms experienced in the previous week in the participants included in the study was 3.7%.5 In another study that investigated the psychological effects of the SARS epidemic, the prevalence of symptoms of depression, anxiety, and post-traumatic stress disorders was reported to be between 10% to 18%.22 In a survey-study conducted during the initial phase of COVID-19 pandemic, it was reported that 28.8% of the participants showed moderate to severe anxiety symptoms and 16.5% moderate to severe depressive symptoms.10 Female gender, student status, the presence of specific physical symptoms such as myalgia, and dizziness were associated with higher levels of stress, anxiety, and depression.10During the pandemic process, the severity of containment and closure measures should be taken into account at the point in time for each participant’s assessments. In this context, the Oxford COVID-19 Government Response Tracker (OxCGRT), a dataset, constantly updated, expanding, and easy to use with global scope provide to detection of COVID-19 cases, deaths, and their effects on economic and social welfare [21]. Anxiety and depression have been reported as common psychological problems during crisis periods in pandemics. Higher depressive levels were found in individuals with quarantined or indirectly exposed to SARS in a study to investigate the psychological effects of the SARS epidemic in 2003.23 There has been a significant increase in suspected depression after the COVID-19 pandemic. In a study, depression was reported as 10% in RA patients during the COVID-19 pandemic.24 While the definite anxiety rate was approximately 9% in RA patients before the pandemic, this rate increased to 12% in 2020 during the pandemic period.24 In another study investigating the psychological effects of the pandemic on patients with rheumatic diseases, the frequency of anxiety was found to be 20%, depression 43%, and post-traumatic stress 28%.25 Female gender, having a lower education level, having children, and living in a large family are independent risk factors associated with anxiety and depression in patients with the rheumatic disease.25 RA patients are more likely to experience anxiety symptoms due to higher infection risk than the healthy population.22 The confusing information about the benefits and harms of rheumatic drugs against COVID-19 may contribute to this situation.24 In our study, RA patients treated with conventional synthetic DMARDs had mild to severe anxiety symptoms in 46 (65.7%) and mild to severe depression symptoms in 42 (60%) at the first visit, based on the Beck Depression Inventory. At the second visit, 38  of the patients showed depressive symptoms. The mean Beck Anxiety Inventory score and Beck Depression Inventory score were decreased at the second visit compared to the first visit for both conventional synthetic and biological DMARDs group. However, no significant differences were found in scores between visits. In addition, there were no statistically significant differences in the Beck Anxiety Inventory and the Beck Depression Inventory scores in the comparisons of all patients for gender, education, marital, working, and economic status.The prevalence of depression in RA patients was reported to be between 14.8% and 38.8% according to different measurement methods.26 Discontinuation of the drugs may cause increased disease activity, morbidity, and mortality. In a study, it was determined that 22.4% of the patients with rheumatic diseases discontinued or stopped their medications during the COVID-19 pandemic. And most of them were biological DMARDs such as anti-IL-1/anti-TNF agents, tocilizumab, and rituximab.25There has been occurred a concern associated with the use of immunosuppressive drugs in patients with the rheumatological disease with the COVID-19 pandemic. EULAR recommends that patients with rheumatic diseases taking conventional/biological DMARDs, JAK inhibitors, steroids, and methotrexate should not discontinue unless a specific cause.In our study, there was no patient who discontinued both conventional synthetic and biological DMARDs, except for patients with a history of coronavirus. The limitation of the study was that it was a short-term evaluation for the depression and anxiety severity during the COVID-19 pandemic.Based on the results of this study, the severity of anxiety and depression was higher in RA patients taking immunosuppressive therapy during the COVID-19 pandemic compared to the general population. The difference in anxiety and depression symptoms between RA patients receiving conventional synthetic and biological DMARDs does not attain statistical significance. RA patients are likely to experience anxiety and depression during the period of the pandemic. Therefore, it is necessary to be careful in terms of psychological changes and to take precautions if necessary. The anxiety and depression rates have not changed statistically in a short time in RA patients. This may be related to the fact that even during the deepening of the pandemic crisis, according to the recommendations of rheumatology societies and especially EULAR, it was stated that drugs such as conventional/biological DMARDs, JAK inhibitors, steroids, and methotrexate should not be discontinued unless COVID-19 infection. However, studies with longer follow-up periods are needed for long-term data.


A logistic regression model revealed that secundiparas with previous SSCS-F-VD had an association with PTB in the following delivery compared with term SVB, with an OR of 2.756 . Conclusion: Previous SSCS-F-VD is associated with PTB in the following delivery, offering valuable insights for pregnancy management and patient counseling.Background: Studies have found an association between second-stage cesarean sections (SSCSs) and subsequent preterm birth (PTB). We aimed to evaluate if secundiparas with previous second-stage cesarean sections due to a failed vacuum delivery (SSCS-F-VD) are associated with PTB in the subsequent delivery compared with secundiparas with previous spontaneous vaginal birth (SVB) at term. A secondary aim was to compare this association with secundiparas with a previous SSCS at term. Methods: A historical, prospective, longitudinal cohort study was conducted in a large tertiary university hospital between 2006 and 2019. Matched mothers who experienced first and second births at the indexed hospital, excluding those with a previous miscarriage or multiple pregnancy in either the first or second birth were grouped based on the mode of delivery and gestational week of the first birth. Results: Parturients with term SVB and term SSCSs were less likely to experience PTB in the following delivery compared with those who underwent an SSCS-F-VD, with 496/14,551 (3.4%) versus 6/160 (3.8%) versus 5/61 (8.2%), respectively, at  Assisted vacuum delivery is an efficient intervention for a successful vaginal birth in cases of a prolonged second stage, a non-reassuring fetal heart rate, or limited maternal expulsive efforts. Potential neonatal and maternal complications limit its performance to medically indicated cases with appropriate fetal head position and cervical conditions ,2. A faiPrevious studies found an association between a second-stage cesarean sections (SSCSs) and preterm birth (PTB) in the subsequent delivery ,9,10,11.Secundiparas (parity = 2) subsequent to a failed-vacuum delivery represent the extreme of SSCSs, given that the fetal head was impacted in the pelvis prior to the cesarean, and as such, we assume that surgical complications, including uterine incision extension and difficulty locating the low-segment transverse from the cervix occur more often, thus placing this group at increased risk for PTB compared with other indications of SSCSs.The primary aim of this study was to evaluate if secundiparas with a previous second-stage cesarean section due to a failed vacuum delivery (SSCS-F-VD) are associated with PTB in the subsequent delivery compared with secundiparas with a previous SVB at term. The secondary aim was to compare this association with secundiparas with a previous SSCS at term.The study was conducted in a single, large tertiary university medical center, Shaare Zedek Medical Center (SZMC), Jerusalem, which is one of the two largest obstetric centers in the country, between January 2006 and December 2019. The obstetric profile of the study population is characterized by approximately 14,500 annual deliveries, which is estimated to comprise 10% of the national deliveries. Parity characteristics have been relatively stable throughout the last two decades; there have been ~25% nulliparous, ~18% grand multiparous (parity ≥ 6), ~12% cesarean deliveries, and ~5% vacuum deliveries. Over 95% of deliveries are funded by the national public insurance, managed by midwives and residents, and supervised by senior obstetricians.The current study was a historical, prospective, longitudinal cohort study. All matched mothers who experienced a spontaneous onset of labor at first birth (P1) were screened for inclusion and exclusion criteria. Those with a live birth at ≥24 weeks of gestation at P1 and a second birth (P2) at the indexed setting were included. Parturients with a multiple gestation at either first or second birth, women with previous miscarriages, women with a non-vertex SVB, and women with an unplanned CS prior to the second stage of labor were excluded. Gestational weeks were categorized as PTB (<37 weeks) or term (37–42 weeks). Modes of delivery were grouped into five categories: SVB, vacuum delivery, planned CS at term, SSCS, and SSCS-F-VD. Matched mothers were grouped into six categories according to three criteria at P1: gestational week at birth, mode of delivery, and stage of CS: (1) term SVB, (2) term vacuum delivery, (3) term planned CS, (4) term SSCS, (5) term SSCS-F-VD, (6) PTB with either mode of delivery. Induction of labor (IOL) was not a criterion for subgrouping women, as we did not separate between indicated and spontaneous PTB.Based on the obstetric profile of the study population, we expected to have 19,300 matched mothers in the study, of which 792 grouped in the planned CS, SSCS, and SSCS-F-VD groups. This estimation was based on the following: 200,000 births at SZMC during the study period 14,500 × 14), of which ~25% were primiparas, i.e., 50,000. Based on data from our medical center, 33% of primiparas do not return for the second delivery, and they deliver elsewhere [,500 × 14Data were extracted from a computerized database and updated in real time by midwives and obstetricians attending the labor and delivery. Most of the variables (11/12 (92%)) in the medical record were fixed, mandatory fields required to be completed before transferring the parturient to the postpartum ward. Data retrieved for both P1 and P2 included maternal age, gestational age at delivery, onset of labor , mode of delivery , length of second stage at P1 (to classify the groups into term SSCS or term SSCS-F-VD and exclude women with CSs prior reaching full dilatation), newborn birthweight (BW), Apgar score at 1 and 5 min, and admission ward of the newborn.Secundiparas with a history of SSCS-F-VD in their previous delivery were identified by searching the electronic medical database for ‘failed vacuum delivery’, as coded by the International Classification of Diseases (660.7), at P1. Most parturients with a breech presentation in our facility undergo a trial of external cephalic version at 37–38 weeks of gestation, and those who fail undergo a planned CS. Breech vaginal birth is performed under strict criteria. Indications for the CSs were documented for parturients grouped in the planned CS group: breech presentation, placental previa, macrosomia, maternal request, and a non-trial of labor due to a non-reassuring fetal monitor or other reasons.p-value ≤ 0.05 using the Chi-square test or Fisher exact test for categorical variables. The Wilcoxon signed-rank test was employed for ordinal or continuous variables with non-Gaussian distribution, and the Student paired t-test was utilized for continuous variables with a normal distribution. A univariate analysis followed by a binomial logistic regression were utilized with the aim of determining the effect of gestational week and mode of delivery at P1 on the likelihood of PTB at the second birth. Included in the model were gestational age and mode of delivery (P1) and maternal age (P2). Secundiparas with a history of SSCS-F-VD were associated with PTB compared with parturients with a history of SVB at term. Analyses were performed using IBM SPSS® Statistics (version 29).Data were validated by defining distributions and quantifying missing values. Obstetric characteristics, stratified by first and second delivery, were presented as proportion, median, or mean with interquartile range or standard deviation, depending on the variable characteristics: categorical, ordinal, or continuous, respectively. Statistical significance was defined by a two-sided The study protocol and amendments were approved by the Institutional Review Board of the Shaare Zedek Medical Center (IRB # 0100-19). As this was a historical study, a waiver of informed consent was obtained. The manuscript is presented according to the STROBE guidelines .During the 14 years of the study, a total of 91,955 primiparas and secundiparas gave birth at SZMC. After excluding multiple gestations of either primiparas or secundiparas, those who delivered elsewhere at first or second birth, and those with missing data regarding the inclusion and exclusion criteria, a total of 25,275 matched mothers who delivered both first and second births at SZMC remained, for a total of 50,550 births. Further exclusion of those with a previous abortion or who underwent a first-stage CS reduced the study group to 18,902 matched mothers. All six categories are presented in p < 0.001. There was a low incidence of CSs in both the first  and the second  birth, which is possibly explained by the exclusion of cases of term CSs in active labor prior to the second stage . Secundiparas with a previous PTB had a prevalence of 20.4% for experiencing PTB in the second delivery , compared with secundiparas, with 822 (4.3%), nd stage . The predelivery .p < 0.001; explained 6.8% of PTBs at the second delivery; and correctly classified 95.7% of the cases. The calculated area under the ROC curve of the model was 0.653 , p < 0.001  and maternal age (P2). The model included 18,900 cases; was statistically significant, χ2 (6) = 388.422,  < 0.001 .p = 0.031). More importantly, this study highlights that secundiparas with a previous SSCS-F-VD have more than double the prevalence of PTB in the subsequent delivery compared with secundiparas with a previous SSCS for indications other than a failed vacuum delivery, at 5/61 (8.2%) versus 6/160 (3.8%), respectively. This finding suggests that women undergoing an SSCS-F-VD are a unique clinical subgroup among those with an SSCS, necessitating subgroup analysis. This is in contrast to previous studies that focused on the mode of delivery and PTB, categorizing women undergoing an SSCS as a homogenous group without clinical subgroupings [We have shown in this study that secundiparas with a previous SSCS-F-VD are associated with PTB in the subsequent delivery compared with secundiparas with a previous SVB at term, with an OR of 2.756 , which is possibly resultant of excluding the subset of parturients with an SSCS-F-VD from the group, as they have a high association with PTB. Another explanation could be that the association between SSCSs and PTB is correct, yet the sample size was insufficient to show significance. Interestingly, in our study, the group of planned term CSs was found to have an association with PTB compared with term SVB, with an aOR of 1.925 . As this finding was different from others [A systematic review and meta-analysis aimed to study the association between the mode of delivery and PTB in the following delivery and identified 8 retrospective studies which included 10,079,942 women, reporting the aRR for a PTB in the following delivery. Five of the eight studies 62.5%) reported an increased risk of PTB subsequent to prior CDs, with an overall aRR of 1.12  . It is o% reportep = 0.02.Concurrent to the current study, a multicenter retrospective study, including women from the current study, focusing on a similar clinical question was published . That stp < 0.001, implying that there are other factors not included in the model that play a significant role in PTB that need to be uncovered in future studies. However, given that the aim of the study was to evaluate the contribution of each independent variable to the model and not only to discriminate between those who will experience PTB and those who will not, we believe that it is worthwhile to present the results of the model despite its poor discriminatory ability.As PTB is known to be influenced by multiple factors, and given that known factors such as previous PTB, multiple pregnancies, and previous miscarriages were excluded or stratified in the current study, it is not surprising that a poor overall discriminatory ability of the binomial logistic regression model was identified, as seen by the calculated area under the curve (AUC) of the receiver operating characteristic (ROC) of 0.653 , Future studies are needed to look in depth at surgical documentation of lower uterine extensions. This will enable analysis of the association of such extensions with preterm delivery in the following delivery and whether structural cervical damage can explain the association between SSCD and subsequent PTB.The current study design excluded known risk factors for PTB, such as multiple pregnancy and previous miscarriage. The effect of parity was neutralized by including only matched primiparas and secundiparas. Each woman served as her own control, and as such, many of the confounding factors associated with PTB, such as socioeconomic status, BMI, genetic factors, chronic illness, smoking, and alcohol habits, that were not available or missing from the dataset were neutralized. The vast majority of variables analyzed (11/12) are mandatory fields in the electronic medical records and were therefore accessible. Based on the obstetric profile of the study population, we expected to have 19,300 matched mothers in the study, of which 792 grouped into the planned CS, SSCS, or SSCS-F-VD groups. In actuality, we had 18,902 matched mothers, 98% of the expected number, with 716 parturients who underwent a CS at first birth (90% of the estimated number). The similarity between the expected number of parturients to be included in the study and the actual number included strengthens the internal validity of the results, verifying that selection bias was not a significant issue.The current study has several limitations. Factors that are associated with a PTB, such as a urinary tract infection, placental abruption or circumvallate placenta, polyhydramnios, previous cervical conization or LOOP excision, and systemic infections such as COVID or appendicitis, were missing and, therefore, not able to be adjusted for in the regression analysis. Furthermore, grouping IOL with any one of the six subgroups studied without analyzing it as an independent factor could have had a confounding effect on the association studied.Single-center studies are homogenous by nature, unlike multicenter studies that have potential differences in obstetric management and treatment protocols, which could affect the external validity of the study. However, strict inclusion criteria of a narrowly defined group of parturients with similar characteristics that were described by others minimize the significance of this limitation. Further strengthening the external validity of this study was the strong association of PTB among secundiparas with a previous PTB that was similar to the documented literature. The disadvantage of a small sample size leads to the possibility of the study being underpowered to show a significant association.The current study shows that secundiparas with a previous SSCS-F-VD are associated with PTB compared with secundiparas with a term SVB or an SSCS for an indication other than a failed vacuum delivery. This finding suggests that SSCS-F VD are a unique clinical subgroup among SSCSs. Thus, future studies should consider addressing this group as an independent subgroup of SSCSs, offering valuable insights for subsequent pregnancy management and patient counseling following an SSCS-F-VD.


The human gastrointestinal mucosa is colonized by thousands of microorganisms, which participate in a variety of physiological functions. Intestinal dysbiosis is closely associated with the pathogenesis of several human diseases. Innate lymphoid cells (ILCs), which include NK cells, ILC1s, ILC2s, ILC3s and LTi cells, are a type of innate immune cells. They are enriched in the mucosal tissues of the body, and have recently received extensive attention. The gut microbiota and its metabolites play important roles in various intestinal mucosal diseases, such as inflammatory bowel disease (IBD), allergic disease, and cancer. Therefore, studies on ILCs and their interaction with the gut microbiota have great clinical significance owing to their potential for identifying pharmacotherapy targets for multiple related diseases. This review expounds on the progress in research on ILCs differentiation and development, the biological functions of the intestinal microbiota, and its interaction with ILCs in disease conditions in order to provide novel ideas for disease treatment in the future. In the absence of TLR11, immune responses to parasites is depend on the indirect stimulation of DCs provided by commensal microbes. Gut bacteria will activate MyD88 via TLR2, TLR4, and TLR9 to generate IL-12 which could response to the parasite . Moreover, it showed that the supplementation of Lactobacillus reuteri or transplantation with fecal bacteria from wild-type mice had significant anti-tumor effects in HCC mice . The gut is an organ with the largest surface area and mucosal interface , 32, and22.1From 2009 to 2010, lymphocyte-like non-T cells, non-B cells, and natural killer (NK) cells were described in several papers –47. The ILCs are derived from common lymphoid progenitors (CLPs) in the bone marrow of central immune organs  and dispILCs are innate immune cells with adaptive immune function . Their sin vitro transformation of ILC3s, ILC2s, and ILC1s is reversible. For example, human ILC3s are converted into ILC1s under the action of IL-12. Meanwhile, this process can be reversed by IL-1β and IL-23 and accelerated by the vitamin A metabolite retinoic acid , T cell factor 1 (TCF1), and GATA-3 , 86–88 a2.2.3via antigen-specific RORγt+ Treg cells and Th17 cells   induced colitis mice can increase the production of ILC3s and IL-22 in the mice colon, which has protective effects on intestinal injury . SCFAs are not just the body’s energy source , but alsin ILC3s . Ligandsfunction , 165. SC (FFAR2) , 167. Inl injury . In GF ol injury , on the eriments . In addiammation .3.23.2.1Toxoplasma gondii can induce the transition of NK cells to the ILC1s-like phenotype (Eomes CD49a Ly6C). The gut microbiota interacts with NK cells, such as feeding mice a high-salt diet can increase the abundance of Bifidobacterium in the intestine, resulting in increased intestinal permeability and localization of Bifidobacterium within the tumor, thus enhancing NK cells function and tumor regression, and injecting Bifidobacterium into tumors can activate NK cells and inhibit tumor growth , higher concentrations of pro-inflammatory bacteria were also found in a mouse model of inflammatory bowel disease, especially increased Akkermansia muciniphila, which can degrade mucus  in the liver and spleen, causing systemic inflammation  and ulcerative colitis (UC) , 188. IBpatients , 191, anpatients . In the patients .Escherichia coli adhering to the intestinal mucosa of IBD patients can enhance the production of IL-22 by ILC3s , and environmental allergy, have become important public health problems worldwide . FA is apeptides , 134. ILe models , 217. Reoduction .However, ligands produced from tryptophan, a metabolite obtained from the diet and bacterial metabolism, interact with AHR on the surface of ILC3s. This can also stimulate the production of IL-22 to modulate the permeability of the epithelial barrier . If the via direct and indirect pathways. The changes in the composition and proportion of gut microbiota communities can cause skin barrier dysfunction and immune system disorders, which are the key pathophysiological mechanisms of AD development. Tryptophan is is a metabolite of the gut microbiota, it act as ligands that can activate the AHR on ILCs to induce IL-22 secretion, driving the release of antimicrobial peptides (AMPs) and providing protection from infections by pathogens (Lactobacillus and Bifidobacterium produce gamma-aminobutyric acid (GABA), which could be used by inhibitory interneurons as neurotransmitters to inhibit pruritogenic response neurons. Scratching also stimulates ascending nociceptive neurons that project to the supraspinal structures, which in turn connect directly or indirectly to descending modulatory pathways to inhibit pruritogenic response neurons, relieving itching  FA mice models, oral administration of OVA or peanuts can lead to type 2 inflammation, diarrhea and anaphylactic symptoms such as body temperature decrease is of FA . Anothersymptoms . The skiA models . This suA models , 226. BuA models . These dA models Figure 34.3-/- mice exhibit a susceptible to colon cancer, IL-22 plays a key role of in controlling tumorigenesis and epithelial cell proliferation in the colon. However, Patients with IBD have a considerably elevated risk of CRC, IL-22 can activate STAT3 in IECs to promote cell proliferation and play a major role in maintaining tumor development  through IL-22, which has pro-inflammatory effects and can further exacerbate intestinal inflammation  in order to promotes the occurrence and development of cecum lesions, and exacerbating inflammatory reactions  in C57BL/6 mice, it was shown that IL-33 can drive the expansion of ILC2s that produce type 2 cytokines  and reduce the inflammatory mediator IFN-γ, IL-12 and TNF-α, leading to the production of anti-inflammatory M2 macrophages. This, in turn, amplifies Foxp3t of ECM . Cryptosed water . This paporidium . Eimerial mucosa . The reseactions . It suggype mice  (Figure5in vivo is reversible needs to be examined. Moreover, the differences between the subgroups produced during traditional developmental processes need to be clarified further. The two-way regulatory mechanism of ILCs in the body needs to be elucidated to explore the correlation between ILCs and various diseases and to provide new strategies for disease diagnosis and treatment. Therefore, comprehensive analysis methods are required to study the interaction between ILCs and the intestinal microbiota, and this could become a key topic for the treatment of clinical diseases in the future. Innovative ILC-based interventions should be explored to better understand the relationship between ILCs and diseases and achieve a more comprehensive understanding. This could help in safeguarding the health of the human race.In recent years, ILCs have become a research hotspot in the field of immunology and cell biology. Despite their small percentage, these lymphocytes provide protection against bacteria, parasites, and viruses, and also play a role in tissue repair and regeneration in the mucosa. However, ILCs are bidirectional, and their inflammatory effects on the body can aggravate intestinal inflammation and even cause cancer. ILCs are characterized by their ability to induce corresponding cytokines on the surface of the intestinal barrier to achieve rapid responses. Therefore, how the interaction between ILCs and the intestinal microbiota can be equilibrated and how internal homeostasis with the microbiota can be maintained to ensure that the cytokines secreted by ILCs play a protective role are worthy of further exploration. Nevertheless, some issues related to ILCs remain to be addressed. For example, whether the process of ILC trans-differentiation YG did the literature review and wrote the main body of the article. YiL and ML co-supervised on finishing this review. YL, BR, ZL, XN constructed the idea.ML made valuable comments to the manuscript. All authors contributed to the article and approved the submitted version.


Primary headache disorders can be remarkably disabling and the therapeutic options available are usually limited to medication with a high rate of adverse events. Here, we discuss the mechanism of action of non-invasive vagal nerve stimulation, as well as the findings of the main studies involving patients with primary headaches other than migraine or cluster headache, such as hemicrania continua, paroxysmal hemicrania, cough headache, or short-lasting neuralgiform headache attacks (SUNCT/SUNA), in a narrative analysis. A bibliographical search of low-prevalence disorders such as rare primary headaches retrieves a moderate number of studies, usually underpowered. Headache intensity, severity, and duration showed a clinically significant reduction in the majority, especially those involving indomethacin-responsive headaches. The lack of response of some patients with a similar diagnosis could be due to a different stimulation pattern, technique, or total dose. The use of non-invasive vagal nerve stimulation for the treatment of primary headache disorders represents an excellent option for patients with these debilitating and otherwise refractory conditions, or that cannot tolerate several lines of preventive medication, and should always be considered before contemplating invasive, non-reversible stimulation techniques. Migraine and cluster headache apart, the International Classification of Headache Disorders, 3rd edition (ICHD-3), contains other disabling primary headaches; among these are trigeminal-autonomic cephalalgias, including hemicrania continua and chronic paroxysmal hemicrania, or the short-lasting unilateral neuralgiform headache attacks, and more than a dozen primary headaches classified as “other,” such as cough headache, hypnic headache, or exertional headache  1)..1).A majority of these headaches have a low prevalence rate, and the therapeutic armamentarium is usually limited to pharmacological options with a wide range of side effects, such as indomethacin or antiepileptic medications .Vagal nerve stimulation (VNS) was first investigated in 1990 for the treatment of intractable epilepsy , 4. In tWe performed a narrative review of the literature using PubMed, EMBASE, and Web of Science on September 2022. Keywords included primary headaches 3.2–4.10 from the ICHD-3  was increased in the ipsilateral structures of the left posterior cerebellum and left putamen and in the contralateral areas of the right middle temporal gyrus and right thalamus. Larger increases were seen in the cerebellum and thalamus in the patient with the best clinical response   . Dorsal halamus) . More rehalamus) . At 15 Vhalamus) .An inflammatory component involving cytokines has been suggested to be part of the pathophysiology, outside the central nervous system, of other primary headaches , 36. AssP = 0.008) and ACT2 showed similar results, both in patients with the episodic form, with an efficacy of 48% vs. 6% in the sham group (P < 0.01)  –49. nVNS < 0.01) .Apart from cluster headache and migraine, nVNS has been reported to be effective in other primary headaches, especially indomethacin-sensitive headaches. However, given the rarity of these disorders, the quality of the study is usually power-limited, with a small number of participants given the challenging task to organise a randomised, placebo-controlled study in headache types with low prevalence . IndometThe first study reporting the efficacy of nVNS in other headaches described the effect on participants with hemicrania continua. In 2013, Nesbitt et al. published the test results of two patients who were previously responsive to stimulation of the occipital nerve. Both patients reported an improvement in the background pain and in painful autonomic exacerbations and would recommend the device to other patients . The subTso et al. described the efficacy of VNS in a retrospective description of 14 patients, 8 with hemicrania continua and 6 with chronic paroxysmal hemicrania . Seven oFour patients with hemicrania continua receiving acute pulses summated to 540 s of preventive stimulation per day were assessed by Trimboli et al. Continued treatment for 3 months was beneficial to two patients. One of them presented a 72.7% reduction in headache exacerbations and 1.8/10 in headache severity. However, exacerbations reduced of only 27.3% after 11 months. The second participant had a reduction from 10 to 1 day of exacerbations per month on the third month, which persisted at two monthly exacerbations after 10 months. Headache intensity reduced from 5.9/10 to 2.5/10 on the third month and persisted at 2.9/10 at the end of follow-up .P < 0.05). A 50% improvement in headache frequency was reached between 3 and 6 months of daily treatment. Headache severity also dropped 50%, 2/10 points in a verbal scale, after 3 months, and a further 2/10 points at the end of follow-up (8/10 to 4/10). Similarly, headache duration halved after 3 months. Anxiety and depression scores improved significantly  should be specified. When the option of selecting a dose is available, and there is titration to sensation or muscle response (such as lip pulling), the actual dose selected should be reported . Most stAnother explanation for the disparity of results could be the treatment duration, which was also an important factor in epilepsy studies –67. DescAlso, patients with hemicrania who responded to treatment may need a significantly longer treatment schedule, as compared with those who did not respond in a retrospective cohort, and a higher number of total daily seconds .Adherence could also represent a main issue when assessing nVNS efficacy, as seen in migraine studies . AdherenA smaller and discrete device that uses transcutaneous auricular vagus nerve stimulation has been studied in migraine. The cutaneous distribution of the auricular branch of the vagal nerve is heterogeneous in cadaveric studies . HoweverTreatment with nVNS is well tolerated and efficacious in patients with other primary headache disorders, especially indomethacin-responsive headaches such as cough headache, hemicrania continua, and paroxysmal hemicrania. As with any preventive treatment, nVNS should be trialled for at least 3 months to consider any lack of efficacy, and assessment of adherence and correctness of the technique of stimulation should be granted before withdrawal. Training should be offered and the technique assessed in unresponsive patients. Tolerability could be inversely related to sensitisation in patients with a concomitant migrainous background. A more standardised way of reporting stimulation parameters is desirable for future studies.