Further, high SO2 and low O3 were significantly associated with low MMSE G1 scores. Low O3, high CO, high SO2, high NO2, and high NOx were significantly associated with low MMSE G4 scores, and high PM2.5, high particulate matter with an aerodynamic diameter of ≤10 μm (PM10), high SO2, high NO2, and high NOx were significantly associated with low MMSE G5 scores. Our results showed that exposure to different air pollutants may lead to general cognitive decline and impairment of specific domains of cognitive functioning, and O3 may be a protective factor. These findings may be helpful in the development of policies regarding the regulation of air pollution.The issue of air pollution is gaining increasing attention worldwide, and mounting evidence has shown an association between air pollution and cognitive decline. The aim of this study was to investigate the relationships between air pollutants and cognitive impairment using the Mini-Mental State Exam (MMSE) and its sub-domains. In this study, we used data from the Taiwan Biobank combined with detailed daily data on air pollution. Cognitive function was assessed using the MMSE and its five subgroups of cognitive functioning. After multivariable linear regression analysis, a high level of particulate matter with an aerodynamic diameter of ≤2.5 μm (PM Air pollution has become a serious concern worldwide. The World Health Organization (WHO) estimates that only one in ten people breathe air that does not contain pollutants exceeding the recommended limits. In the Asia-Pacific region, including Taiwan, air pollution is substantially more severe than in most developed countries, mostly due to rapid urbanization and industrialization with fewer emission regulations . Air pol3). Experimental research has demonstrated that PM exposure can lead to diffuse accumulation of cerebral beta amyloid plaque, hyperphosphorylated tau pre-tangles, and peripheral systemic inflammation, which further result in the activation of microglial and astrocytes in the central nervous system (CNS). Moreover, neuroinflammation has been implicated as an important pathway in neurodegenerative disease [3, per se, is a strong oxidative pollutant, which elicits neurological injury by induction of release of free radicals, activation the generation of inflammatory cytokines and compromising the integrity of the blood–brain barrier. Despite most evidence showing its detrimental influence on cognition, a few epidemiological studies revealed protective effects at low concentrations of O3 exposure [2), nitric oxide (NO), nitrogen dioxide (NO2), and nitrogen oxide (NOx) [Various tools are available to measure cognitive function, such as the General Practitioner Assessment of Cognition, Mini-Cog™, and Mini-Mental State Exam (MMSE). Of these, the MMSE is a widely accepted tool that is easy to administer and divided into five subgroups to evaluate different aspects of cognitive functioning . Most pr disease . O3, perexposure . Howeverde (NOx) ,8, and tde (NOx) .In this study, we combined data from two databases, the Taiwan Biobank (TWB) and Taiwan Air Quality Monitoring Database (TAQMD). The TWB included 5000 registered individuals, and the TAQMD was comprised of detailed daily data on air pollution in Taiwan. Choe YM et al. evaluated the usefulness of subscores on the MMSE for predicting the progression of Alzheimer’s disease dementia in individuals with mild cognitive impairment. They found that MMSE memory, orientation, and construction subscores, which are simple and readily available clinical measures, could provide useful information to predict Alzheimer’s disease dementia progression in individuals with mild cognitive impairment, in practical clinical settings . TherefoThe Institutional Review Board on Biomedical Science Research, Academia Sinica, Taiwan, and the Ethics and Governance Council of the TWB, Taiwan granted ethical approval to the TWB. Each participant provided written informed consent, and the study was conducted according to institutional guidelines and the principles of the Declaration of Helsinki. In addition, this study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-E(I)-20180242).2), were recorded. The participants were also asked to complete questionnaires about personal and lifestyle factors in face-to-face interviews with TWB researchers.The TWB was established to record genomic and lifestyle data of Taiwanese residents, and it is currently the largest government-supported biobank in Taiwan ,11. The Demographic data (age and sex), smoking history, medical history (diabetes mellitus (DM) and hypertension), examination findings (systolic (SBP) and diastolic blood pressures (DBP)) and laboratory data  and uric acid) were recorded at baseline. EGFR was calculated using the four-variable Modification of Diet in Renal Disease equation .We assessed the cognitive function of the subjects using the MMSE . The par2.5), PM with an aerodynamic diameter of ≤10 μm (PM10), O3, CO, SO2, NO, NO2, and NOx during each year were calculated, and average yearly data were determined in three steps: (1) the corresponding longitude and latitude of the address were obtained using Google geocoding; (2) the nearest air quality monitoring station was identified from an interpolation point; (3) data from the this station were filtered from the survey date to the previous year, and the average of each air pollution metric was calculated.The TAQMD was established by the Taiwan Environmental Protection Administration, Executive Yuan, and includes data on daily air pollutant concentrations at 74 ambient air quality monitoring stations around Taiwan. In this study, we linked the TAQMD and TWB by the location of the air quality monitoring stations and the area of residence of the participants. The residential address of each participant was used to estimate exposure to outdoor air pollution. The average concentrations of air pollutants, including PM with an aerodynamic diameter of ≤2.5 μm .Data were expressed as mean ± standard deviation, frequency (%), or median (25th–75th percentile) for triglycerides. An MMSE cut-off score of 24 was used to classify the severity of cognitive impairment. Differences between groups were analyzed using the chi-square test for categorical variables and the independent t test for continuous variables. Linear regression analysis was used to identify associations between each air pollutant and MMSE and subscales. A 3, lower total MMSE score, and lower score on each MMSE subscale.The mean age of the 1054 participants was 64.0 ± 2.9 years, and included 519 males and 535 females. The participants were stratified into two groups according to an MMSE score ≥24  or <24 . Comparisons of the clinical characteristics between these two groups are shown in p = 0.009), female , low education years , high BMI , low O3 , and high SO2  were associated with low total MMSE score.2.5 , low O3 , high CO , high SO2 , high NO , high NO2 , and high NOx  were significantly associated with low total MMSE score, whereas PM10 was not significantly associated with total MMSE score.2 , and low O3  were significantly associated with a low MMSE G1 score.3 , high CO , high SO2 , high NO2  and high NOx  were significantly associated with a low MMSE G4 score.2.5 , high PM10 , high SO2 , high NO2 , and high NOx  were significantly associated with a low MMSE G5 score.3 , which was associated with a low MMSE G3 score.We also analyzed correlations between air pollutants and MMSE G2 (registration) and G3  scores. However, no air pollutants were significantly associated with these scores, except for low O2.5, CO, SO2, NO, NO2, and NOx, and low O3 were correlated with low total MMSE score. We further found that these pollutants were separately correlated with different MMSE subscores.In this study of 1054 participants registered in the TWB from around Taiwan, we observed that high PM2.5 was associated with low total MMSE and MMSE G5 (language) scores, and that high PM10 was also related with a low MMSE G5 score. Increasing epidemiological evidence has shown the effect of PM2.5 on worsening cognitive function [2.5 on worsening cognitive function [2.5 could also cause deficits in multiple domains of cognitive function, including executive, memory, visuospatial, and language functions in young adults [2.5 accelerates the accumulation of cerebral β-amyloid and tau hyper-phosphorylation [2.5 and smaller hippocampal volume [2.5 is a risk for the development of cognitive decline and CNS damage. However, results of the association between cognitive decline and the coarser particles of PM10, with an aerodynamic diameter of 2.5 to 10 mm, have been inconsistent [2.5 and PM10 is the ability to more readily cross the BBB and cause more systematic inflammation [10, can reach the CNS via numerous routes, including inhalation through the lungs, direct capture by the olfactory nerve, digestion into the gastrointestinal tract, or cutaneous pathways [The first important finding of this study is that high PMfunction ,14,15,16function . In addig adults . The prig adults ; howeverg adults ,21,22. Pg adults . With rerylation ,25, whicl volume . Taken tnsistent ,28,29,303 was associated with high total MMSE score and G1, G3, G4 subscores, implying the possible neuroprotective effect of O3 on cognitive function. Previous experimental and epidemiological studies have identified that O3, a strong oxidative pollutant and, therefore, potentially toxic to humans, has detrimental effects on the CNS [3 not only directly damages lung function, but also cause systemic effects via the generation of free radicals. Oxidative stress then further increases the expression of inducible NO and subsequent cellular response, which leads to increases in leukotrienes, prostaglandins, tumor necrosis factor, interleukin-1, interleukin-6, and interleukin-8. Through the active transport of endothelial cells adjacent to the BBB or via other proposed methods of entry into the CNS, these molecules can affect astrocytes and then compromise the integrity of the BBB, and this process can be accelerated by free radicals caused by exposure to O3. This vicious cycle has been suggested to lead to the development of neurological disorders and cognitive impairment [3 and a variety of measures for cognition including MMSE Korea child scores, digit-forward span scores, word list recall, recall storage, and frontal assessment battery scores [3 exposure. Although exposure to toxic levels of O3 as an air pollutant has been considered to induce systemic inflammation and damage, exposure to low O3 concentrations has been used for medical purposes via activating Nrf2, which can enhance the activity of antioxidant enzymes such as superoxide dismutase and catalase [3 has a positive effect on the CNS in contrast to the other reported detrimental effects. Further experimental and epidemiological studies are needed to investigate the relationships between O3 as an air pollutant and the CNS to fill this gap in knowledge.The second important finding of this study is that high O the CNS ,14,18,30 the CNS ,31. Inhapairment . Howevery scores . Similary scores . The discatalase . This maThe third important finding of this study is that high CO was correlated with low total MMSE score and recall domain scores, suggesting the detrimental effect on cognitive function. Chang et al. demonstrated the negative effect of CO on cognitive performance , and Shi2 and global cognitive decline and impairment of specific functioning domains including orientation, recall, and language, suggesting its adverse influence on neurocognitive performance. A few studies have investigated the relationships between atmospheric SO2 and neurological dysfunction [2 on the cardiovascular system [2 exposure has been reported, and this may play a role in cognitive impairment [2.5 and SO2 has been reported to result in neurodegeneration, including neuronal apoptosis, and decreased synaptic structural and functional proteins [2 was the one air pollutant related to most impaired trajectories of cognitive functioning according to MMSE subscales, indicating that further studies are warranted to investigate its potential damage on the CNS.In this study, we found negative associations between SOfunction ,41. Sever system ,43, howepairment . Furtherproteins . However2, and NOx were associated with low total MMSE scores, and we further found that NO2 and NOx were associated with low MMSE G4 and G5 subscores. The neurotoxicity of NO as an air pollutant remains unclear. The physiological regulatory functions of endogenous NO include the modulation of blood vessel tone, immune response to neurotransmission and synaptic plasticity, and an excess of NO has been proven to be toxic [2 can negatively affect cognitive function in different periods of life [3 increase in NO2 was associated with a -0.29 point decrease in mental score in two-year-old children, which may suggest its adverse effect on cognitive development [2 was also associated with an increased incidence of dementia in a cohort study of participants aged from 55 to 85 years [2 is a reactive oxidative species, the mechanism by which it damages the CNS can be assumed to involve oxidative stress, to which the brain is especially susceptible [2 exposure could induce morphological changes of the mitochondria in the cortices of rats and interfere with mitochondrial metabolism including increased generation of reactive oxygen species, which in turn affected mitochondrial energy production and biogenesis [An additional important finding of this study is that high NO, NObe toxic . In addibe toxic ,48. NO cbe toxic ,50. Many of life ,16. Durielopment . NO2 wasceptible . A previogenesis . These sThere are several limitations to this study. First, we believe that the relationship between air pollutants and cognitive decline takes a period of time, not a short period of time. However, it is a cross-sectional study, and, therefore, we could not examine the effects of long-term exposure to these air pollutants on cognitive functioning. Follow-up studies are needed to confirm our results. Second, cognitive performance was assessed using only the MMSE and its subscales in our study. MMSE is a screening tool, and it only provides information about cognitive status. As a screening test, it is validated using its total score. Although different items use different cognitive functions, and some of them are summed in subscores, they are not actually sub-scales without psychometric information. Finally, levels of air pollutants were estimated at the home addresses of the participants, which is a crude measure of exposure to air pollution. Although outdoor air pollution is known to substantially contribute to indoor/personal exposure, it may not accurately reflect actual personal exposure; however, we did not have additional information about the air quality indoors.In conclusion, the results of this study showed that exposure to different air pollutants may result in general cognitive decline and impairment of specific domains of cognitive functioning. In addition, we found that most known air pollutants are associated with cognitive decline, and this may be helpful for the government in the development of policies for early diagnosis and long-term follow-up of neurological deficits of people residing in polluted areas.


Ulcerative colitis (UC) is a refractory intestinal disease prone to recurrent attacks, with a high rate of canceration, which seriously affects life treatment. Routine treatment has disadvantages such as long course of treatment, high cost, easy recurrence and limited effectiveness. Clinical practice shows that acupoint catgut embedding therapy has certain therapeutic advantages but lacks evidence of evidence-based medicine. The purpose of this study is to systematically study the effectiveness and safety of acupoint catgut embedding for ulcerative colitis.Retrieve English database  and Chinese database  by computer, and manually retrieve Baidu and Google Academy for randomized controlled trials (RCTs) of acupoint catgut embedding therapy for ulcerative colitis from the time of construction of database to September 2020. Two researchers independently extracted data and evaluated the quality of the literature included in the study, and used RevMan 5.3 software for meta-analysis of the included literature.The study evaluated the effectiveness and safety of acupoint catgut embedding for ulcerative colitis through efficiency, symptom score, colonoscopy score, mucosal healing rate, recurrence rate, incidence of adverse reactions, etc.This study will provide reliable evidence-based evidence for clinical application of acupoint catgut embedding therapy for ulcerative colitis.DOI 10.17605/ OSF.IO / 7T4QV. The clinical manifestations are abdominal pain, diarrhea, mucopurulent, and bloody stools, etc., which are characterized by recurrence and remission. Colorectal cancer is an important complication of long-term ulcerative colitis, with a high mortality. At present, conventional treatment drugs include aminosalicylic acid, adrenocortical hormones, immunosuppressive agents, new immunomodulators, anticoagulants, microbial agents, etc. However, long-term use of these drugs reduces their efficacy and is usually accompanied by variable side effects, such as diarrhea, nausea, vomiting, headache, and osteoporosis, so new treatment options are urgently needed. At present, more and more TCM treatment schemes are applied to the treatment of ulcerative colitis.Ulcerative colitis (UC) refers to a chronic inflammatory disease involving the rectum and colonic mucosa, which is a lifelong disease caused by the interaction of genetic and environmental factors.–11 with significant efficacy, low cost, low adverse reactions, and other advantages. Clinical studies have confirmed that the immune level of patients with ulcerative colitis is lower than that of normal people, while clinical observation has found that acupoint catgut embedding therapy for ulcerative colitis has the effect of reducing inflammatory response, improving immunity, promoting mucosal regeneration, and has definite curative effect. At present, a number of randomized controlled studies have also confirmed this viewpoint,–16 but there are differences in research programs and efficacy of each clinical trial, resulting in uneven research results, which to some extent affects the promotion of this treatment. Therefore, this research plan systematically evaluates the efficacy and safety of acupoint catgut embedding in the treatment of ulcerative colitis, and provides reliable reference basis for clinical application of acupoint catgut embedding in the treatment of ulcerative colitis.Acupoint catgut embedding therapy is a traditional external treatment method in China, which has been widely used in the treatment of hypertension, diabetes, anal fissure, acute and chronic pain, and other diseases,22.1This protocol of systematic review and meta-analysis has been drafted under the guidance of the preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P). Moreover, it has been registered on open science framework (OSF) on September 8, 2020. (Registration number: DOI 10.17605 / OSF.IO / 7T4QV).2.2Since this is a protocol with no patient recruitment and personal information collection, the approval of the ethics committee is not required.2.32.3.1We will collect all available randomized controlled trials (RCTs) on acupoint catgut embedding for ulcerative colitis, regardless of blinding, publication status, region, but Language will be restricted to China and English.2.3.2), regardless of nationality, race, age, sex, course of disease, etc.Patients with definite diagnosis of ulcerative colitis  Primary outcome:1. the total effective rate = (number of complete remission + effective number)/total number ∗100%. Among them, complete remission represents the disappearance of clinical symptoms, and the mucosa is found to be roughly normal by colonoscopy reexamination;the overall effective rate, referring to the diagnostic criteria of ulcerative colitis in the Consensus Opinions on Diagnosis and Treatment Standards of Inflammatory Bowel Disease in China published by the Chinese Society of Traditional Chinese Medicine in 2007,2.effective represents the basic disappearance of clinical symptoms, mild inflammation of mucosa or pseudopolyp formation by colonoscopy reexamination;3.ineffective represents no improvement of clinical symptoms, endoscopy, and pathological examination results after treatment.(2) Secondary outcomes:1.;symptom score2.);colonoscopic score (refer to Baron endoscopy score3.);mucosal healing rate ;5.recurrence rate;6.incidence of adverse reactions.2.41.Repeated published papers;2.Papers whose publications are abstracts or whose data is incomplete and the complete data cannot be obtained after contacting the authors;3.Studies with obvious data errors;4.;Literatures assessed as high risk of bias by randomization or allocation of concealment5.Studies in which treatment groups with other TCM therapies, such as acupuncture, moxibustion, traditional Chinese medicine compound, etc.6.Literatures without relevant outcome indicators.2.5Chinese databases were retrieved using “acupoint catgut embedding”(xue wei mai xian) and “ulcerative colitis”(kui yang xing jie chang yan) as Chinese search terms, including China National Knowledge Infrastructure (CNKI), Wanfang Data Knowledge Service Platform, VIP Database for Chinese Technical Periodicals (VIP), and China Biomedical Database. The English databases, including PubMed, EMBASE, Web of Science, the Cochrane Library, were retrieved with the English search terms of “acupoint catgut-embedding therapy”, “Acupuncture point catgut embedding”, “Ulcerative colitis”, “Colitis Gravis”. In addition, manual searches were conducted in Baidu and Google Academy. The retrieval time was from the establishment of the database to September 2020, during which all the domestic and foreign literatures on the treatment of ulcerative colitis by acupoint catgut embedding were collected. Taking PubMed as an example, the retrieval strategy is shown in Table 2.6Referring to the method of research selection in version 5.0 of the Cochrane collaboration Network system Evaluator Manual, according to the PRISMA flow chart, the 2 researchers used the EndNote X9 document management software to independently screen and check the literature according to the above inclusion and exclusion criteria, and check each other, if there were different opinions, negotiate with a third party to resolve the differences. At the same time, Excel 2013 was used to extract relevant information, including:1.Clinical studies ;2.Interventions ;3.Risk bias assessment elements in randomized controlled trials;4.Outcome indicators.The literature screening process is shown in Figure 2.7Use the Cochrane collaborations tool for assessing risk of bias to do the risk of bias assessment of included studies. According to the performance of the included literatures in the above evaluation items, 2 researchers will give judgments like low risk, unclear or high risk judgments one by one, and cross-check after completion respectively. In case of any disagreement, discussion will be carried out. If no agreement can be reached between the 2, discussion will be made with the researchers in the third party.2.82.8.1The RevMan 5.3 software provided by the Cochrane Collaboration will be used for statistical analysis.1.Relative risk (RR) is selected as the statistic for the dichotomous variable. For continuous variables, weighted mean difference (WMD) is selected when the tools and units of measurement indicators are the same, standardized mean difference (SMD) is selected with different tools or units of measurement, and all the above are represented by effect value and 95% confidence interval (CI).2.P ≥ .1, there is no inter-study heterogeneity; If P < .1, it indicate inter-study heterogeneity. At the same time, I2 value is used to quantitatively evaluate the inter-study heterogeneity. If I2 ≤ 50%, the heterogeneity is considered to be good, and the fixed-effect model is adopted. If I2 > 50%, it is considered to be significant heterogeneity, the source of heterogeneity will be explored through subgroup analysis or sensitivity analysis. If there is no obvious clinical or methodological heterogeneity, it will be considered as statistical heterogeneity, and the random-effect model will be used for analysis. Descriptive analysis will be used if there is significant clinical heterogeneity between the 2 groups and subgroup analysis is not available.Heterogeneity test: Q test is used to qualitatively determine inter-study heterogeneity. If 2.8.2If there is missing data in the article, contact the author via email for additional information. If the author cannot be contacted, or the author has lost relevant data, descriptive analysis will be conducted instead of meta-analysis.2.8.3Subgroup analysis can be carried out according to the condition, which can be divided into active period and remission period; subgroup analysis can be carried out according to the course of treatment; subgroup analysis can be carried out according to the different drug treatment regimens in the control group.2.8.4In order to test the stability of meta-analysis results of indicators, a one-by-one elimination method will be adopted for sensitivity analysis.2.8.5Funnel plots were used to assess publication bias if no fewer than 10 studies were included in an outcome measure. Moreover, Eggers and Beggs test were used for the evaluation of potential publication bias.2.8.6The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) will be used to assess the quality of evidence. It contains 5 domains . And the quality of evidence will be rated as high, moderate, low, and very low.3 The pathogenesis of the disease is still unclear in modern medicine, which may be a complex result of intestinal mucosal immune dysfunction caused by genetic, environmental, infectious, psychological, intestinal mucosal barrier function defects and other factors. Although there are many treatment options at present, the curative effect is limited and the condition is repeated. The patients should not only endure the pain of the disease itself, but also suffer from various adverse reactions of treatment, which seriously disturb the physical and mental health of the patients.Ulcerative colitis belongs to the category of “Chang pi”(hematochezia), “Li ji”(dysentery), “Chang feng”(hematochezia), “Zang du”, and “Xia li”(diarrhea) in TCM. Its basic pathogenesis is heat and humidity accumulating in the intestines and qi stagnation and obstruction of collaterals. ST37(Shangjuxu), ST25(Tianshu), and BL25(Dachangshu) can regulate the expression of IL-17, β2AR and NF-KBp65, and play an anti-inflammatory and mucosal repair role.Acupoint catgut embedding therapy is the extension and development of acupuncture and moxibustion therapy. Under the guidance of meridian theory, catgut or absorbable surgical thread is implanted into corresponding acupoints to produce a lasting, slow and stable acupuncture effect during the process of decomposition and absorption. Commonly used acupoints include ST36(Zusanli), ST25(Tianshu), BL25(Dachangshu), RN12(Zhongwan), BL20(Pishu), ST37(Shangjuxu), etc. ST36(Zusanli) has the effect of reinforcing the vital essence and strengthening the primordial qi, as well as enhancing immunity; ST25(Tianshu) has the effect of regulating qi and strengthening spleen, and relieving diarrhea with astringents; RN12(Zhongwan) has the effect of strengthening spleen and regulating stomach, and regulating qi; BL20(Pishu) has the effect of strengthening spleen, removing dampness and anti-diarrhea; BL25(Dachangshu) and ST37(Shangjuxu) have the effect of promoting qi and activating blood circulation, and keeping balance of yin and yang. Clinically, acupoint selection according to syndrome differentiation can achieve the functions of dredging meridians and collaterals, regulating qi and blood, and adjusting viscera. Modern studies have confirmed that ST36(Zusanli) and ST37(Shangjuxu) have the functions of regulating immunity and gastrointestinal smooth muscle movement, improving intestinal blood flow, promoting the neogenesis of diseased tissues and repairing ulcers,Clinical studies have confirmed the efficacy of acupoint catgut embedding in the treatment of ulcerative colitis is reliable, however, this is not consistent with the evidence from RCTs. With the increasing number of clinical trials, it is urgent to systematically evaluate the efficacy of acupoint catgut embedding in the treatment of ulcerative colitis. In this study, we will summarize the latest evidence of the efficacy of acupoint catgut embedding in the treatment of ulcerative colitis. This work also provides useful evidence for determining whether acupoint catgut embedding therapy is effective and safe for patients with ulcerative colitis, which is beneficial to both clinical practice and health-related decision makers.However, this systematic review has some limitations. There may be some clinical heterogeneity among the included studies in terms of acupoints, the material of embedding thread, and the degree of patients illness. In addition, the course of treatment varies among patients, which may have an impact on the outcome. Due to the limitation of language ability, we only search English and Chinese literature, and may ignore studies or reports in other languages.Data curation: Ming Sheng Chen, Ping Xin.Funding acquisition: Xiangdong Yang.Investigation: Xiangdong Yang.Resources: Kaidi Feng.Software: Tianyu Zhao.Writing – original draft: Ming Sheng Chen, Ping Xin.Writing – review & editing: Xiangdong Yang.


There are high cutting temperatures, large tool wear, and poor tool life in conventional machining, owing to the superior strength and low thermal conductivity of titanium alloy. In this work, ultrasonic atomization assisted turning (UAAT) of Ti6Al4V was performed with a mixed water-soluble oil-based cutting fluid, dispersed into tiny droplets by the high frequency vibration of a piezoelectric crystal. Different cutting speeds and two machining environments, dry and ultrasonic atomization assisted machining, were considered in the investigation of tool life, tool wear morphology, surface roughness, and chip morphology. In comparison with dry machining, UAAT shows lower tool wear and longer tool life due to the advantages of cooling and lubrication. Furthermore, better surface roughness, smoother chip edges, and shorter tool-chip contact length were obtained with UAAT. Titanium alloy has a high strength–weight ratio, good corrosion resistance, outstanding mechanical and thermal properties, and is currently one of the most widely used materials in aerospace, shipbuilding, medical, and other fields ,2,3,4,5.Laser-assisted machining (LAM) is an effective method, which reduces the cutting resistance of the material by locally heating and softening the workpiece . DifficuAlthough LAM has already been used in various fields, there are still some drawbacks. Bejjani et al. found that an explanation of increased tool life with LAM has been related to tool–particle interactions, where the particles displace in the softer matrix instead of breaking and contributing to additional abrasion wear . AdditioHeat is generated at the cutting zone in LAM, and most of the resulting heat remains in the chip. Therefore, it is necessary to introduce a technique with cooling and lubricating effects to assist LAM. Although flood coolant and cryogenic coolant have noticeable abilities to reduce the heat at the cutting zone, they will worsen the softening effect of LAM. Minimum quantity lubrication (MQL) is considered as a potential alternative cooling strategy to conventional flood coolant and cryogenic coolant ,23,24,25UA has been used for inhalation drug delivery, fuel combustion, analytical nebulizers, and air purification . Howeverρ, σT, σY, δ, H, λ, and E are the density, tensile strength, yield strength, elongation, hardness, thermal conductivity, and elastic modulus of Ti6Al4V, respectively. The experiments were carried out on CKD6136i made by Dalian machine tool Group Co. Ltd. . The schematic diagram of the computerized numerical control (CNC) turning lathe is shown in Ti6Al4V alloy was taken as the workpiece and prepared with a diameter of 60 mm and a length of 300 mm. Its chemical composition is given in Water-soluble oil Huike 206 was dispersed into deionized water with fraction weight of 4 wt %. This type of mixing cutting fluid is environmentally friendly and suitable for industrial applications. An ultrasonic nebulizer  was used in this work, as shown in Two types of turning environments were considered, namely dry machining (DM) and ultrasonic atomization assisted machining (UAAM). The main turning parameters are the cutting speed, feed rate, and cutting depth, as shown in L, using a portable roughness instrument , and the average values were plotted. In addition, EDS of the tools was conducted using energy dispersive X-ray spectroscopy (EDS) spectrums. The cutting distance L of each group test can be calculated by the following Equation (1).v is cutting speed (m/min), l is cutting length (mm), n is rotation rate (r/min), and f is feed rate (mm/r).Scanning electron microscopy  was used to capture the tool wear and chip morphology. The tool wear was measured three times, and the average value was regarded as the final tool wear value. The surface roughness was measured at three different positions of the workpiece after cutting distance VB = 0.3 mm), it is regarded as the tool life criterion [Tool wear and its mechanism have been reported by Li and in other literature ,32,33. Iriterion . Two macL under dry cutting conditions. When the cutting speed is 30 m/min, the friction between the workpiece and the tool surface produces high temperatures. The tool coating falls off, owing to the different thermal expansion coefficients of the coating material and the tool substrate. Falling coating powders gather in the cutting area and result in obvious abrasive wear, as shown in L under UAAT conditions. When the cutting speed is 30 m/min, there is a relatively complete coating on the tool surface under ultrasonic atomization assisted cutting, as shown in 2. According to In order to characterize the mechanisms of tool wear, energy-dispersive X-ray spectroscopy (EDS) was taken from points, as shown in L by each tool. Higher Ra was found under dry machining, owing to the higher adhesion and friction at the chip–tool interface. At the lower cutting speed of 30 m/min, the surface roughness Ra decreased by 15.2% when ultrasonic atomization was applied during machining. At the higher cutting speeds of 40 and 50 m/min, UAAT reduced the surface roughness by 20.1% and 27.4%, respectively. At the highest cutting speed of 60 m/min, a 47.6% decrease in the surface roughness was observed. Obviously, the surface roughness was improved by UAAT at different cutting speed. Increasing the cutting speed, the improvement of surface roughness by UAAT is more significant. Consequently, UAAT brings reduced surface roughness with the increase of the cooling, lubricant effect, and the reduction of tool wear.A novel process of ultrasonic atomization assisted turning (UAAT) was proposed for the machining of Ti6Al4V alloy. A series of turning experiments were conducted in order to investigate the effects of dry and ultrasonic atomization on the cutting performance. The tool life was characterized quantitatively, whereas the tool wear and the chip morphology were analyzed. The following conclusions can be drawn:UAAT has a lubrication effect, which is useful to improve tool life. Under the same processing parameters, the tool life of UAAM was approximately 1.6 times longer than that of dry machining.UAAT can reduce oxidation wear and adhesion wear. Under the same processing parameters, the content of elements such as oxygen and titanium decreased by 70.5% and 3%, respectively, compared with dry cutting.UAAT has also a cooling effect, which helps to reduce the surface roughness of the workpiece. Under the same processing parameters, the surface roughness was reduced by up to 47.6% compared with dry machining.Chip morphology revealed the formation of curved chips under dry machining and straight chips when ultrasonic atomization was employed. Shorter chips were produced by UAAT, with a smaller radius of curvature and smoother edges.


Chronic cough is a common symptom in children and protracted bacterial bronchitis (PBB) is one of the causes of chronic cough. However, the understanding of this disease remains limited. The present study aims to update PBB in children.The clinical data of children with PBB from 2014 to 2018 were retrospectively analyzed, and PBB clinical features of published studies were summarized. Electronic databases were searched in May 2019. Clinical studies were included in the present study. Reviews were undertaken in duplicate.Streptococcus pneumonia, 3 Staphylococcus aureus, and 1 Pseudomonas aeruginosa). Twenty cases were completely remitted after treatment. In the 14 studies, the patients with PBB were typically younger than 3 years old, accompanying wheezing and airway malacia. Co-infection was common in most western cases, Streptococcus pneumonia, Haemophilus influenza and Moraxella catarrhalis were the top three pathogens. Symptoms were improved in most patients, whereas some cases with comorbidities required prolonged antibiotics treatment.Totally 712 cases were analyzed in this study, including 52 cases in our center and 660 cases from 14 studies. In the 52 cases, 88.5% of patients with PBB were less than 6 years old and all of them complained of wet cough. Three cases were confirmed with laryngomalacia, and microbiologically-based-PBB were identified in 13 cases (9 Streptococcus pneumoniae and Haemophilus influenza were the most frequent etiology in China and western countries, respectively. Patients with comorbidities may need extended antibiotics treatment for more than 2 weeks.PBB is common in male infants with chronic wet cough and accompanied by wheezing and airway deformities. Most cases are clinically diagnosed PBB in China and microbiologically-based-PBB is common in western countries. Co-infection could be found,  Protracted bacterial bronchitis (PBB) is an old diagnosis and is considered to be the main cause of chronic wet cough in children , 2. The Patients diagnosed with PBB and underwent follow-up (aged <14 years) at the First Affiliated Hospital of Guangxi Medical University between January 2014 and December 2018 were enrolled in this study.4cfu/ml) in sputum or bronchoalveolar lavage fluid (BALF); cough significantly improved after 2 weeks of antibiotic treatment. (3) Exclusion criteria: patients with mycoplasma infection, infiltrate on chest X-ray or CT, immunodeficiency, congenital heart disease, and other basic diseases.Diagnostic criteria for PPB : (1) cliDemographic characteristics , clinical manifestations , physical examination findings, auxiliary examination , treatment history, and follow-up information were collected for all enrolled patients. Follow-up information included response to antibiotics, duration of cough, complications, and prognosis which were collected by telephone interview or outpatient consulting every 2 weeks, for a total of 4 weeks.We completed a systematic search of databases  in May 2019. Clinical studies with detail data  were included in the present study. Reviews were undertaken in duplicate. We summarized the main observation indexes, including gender, age, course of the disease, pathogenic bacteria, complications, therapeutic drugs, course of antibiotics, and treatment effect.Descriptive analysis was used for this study. Categorical data were expressed as numbers (percentages).A total of 52 children of PBB were enrolled in our center, 33 males and 19 females. Fifteen patients were <1 year, 16 patients were between 1 and 3 years of age, 15 patients were 3–6 years old, 6 patients were 6–14 years old. Most of the patients were younger than 6 years old (88.5%)  was found in nine cases, Staphylococcus aureus (Sa) in three cases, and Pseudomonas aeruginosa (Pae) in one case. Among them, one case of Sa and one case of Spn were found in BALF. Forty-five children received a chest X-ray examination, 27 cases showed increased lung markings. The chest CT of five cases was normal . A total of 14 patients underwent bronchoscopy, and all cases revealed mucosal congestion; 12 cases had thin secretions, 2 cases having purulent secretions. Laryngomalacia was identified in 3 cases, 1 case with tracheal stenosis. Positive bacterial cultures were confirmed in 25% of cases (13/52 cases). All patients received antibiotics treatment for 3–7 days before our treatment, however, symptoms received no improvement or repeated after the withdrawal. In all enrolled patients, 6 patients were treated with oral amoxicillin-clavulanate (Amo) and 15 patients received oral cephalosporin antibiotics. Moreover, 26 patients received intravenous cephalosporin, and 5 children were administrated with Azithromycin (Azi) because they were allergic to penicillin and cephalosporins. All enrolled patients underwent outpatient follow-up every 2 weeks for a total of 4 weeks. Cough in the 24 hospitalized children was improved significantly when discharged from hospital after treatment for 3 to 7 days. Among the 28 outpatients, 20 cases received a resolution of cough after 2 weeks of treatment, while 8 cases still had a cough, including 5 cases with irregular medication and 3 cases accompanied by rhinitis (Table N = 660). We further analyzed the data to confirm whether the clinical feature is similar or not in the east and the west. All of the studies in China were retrospective designs, which showed a high proportion of male and most of them were younger than 3 years old. Fifty two patients performed antibiotic treatment before sputum culture and 178 cases  underwent airway bronchoscopy. The proportion of positive pathogen culture was 63.3% (138/218 cases), other cases were diagnosed as clinically-based-PBB. The detected bacteria were Spn , Haemophilus influenza (Hi) , Moraxella catarrhalis (Mcat) , Sa , Pae , Klebsiella pneumonia , Escherichia coli  and Enterobacter aerogenes . Furthermore, PBB was often accompanied by wheezing and was partially associated with sinusitis, airway stenosis, and tracheobronchomalacia. Most cases improved and some relapsed after treatment with Amo  underwent airway bronchoscopy. Co-infection could be found in the 442 cases of PBB, some patients were caused by multiple pathogenic species and some cases were co-infected by bacteria and viruses. The main pathogens were: Hi , Mcat , Spn , and Sa . Most patients accompanied by wheezing and tracheobronchomalacia. Antibiotic treatment for 2 weeks revealed improvement in most cases, however, 68 relapsed cases  needed to extend the course of antibiotic treatment  and non-Chinese . The Haemophilus influenza in China  and in the western countries  were shown in Fig. Moraxella catarrhalis in Chinese  and non-Chinese cases .The microbiologically-based-PBB and the main pathogens in China and in the western countries were further compared by meta-analysis. The microbiologically-based-PBB in China  and in the western countries  were shown in Fig. .00) were4; 0.74) PBB is also known as persistent endobronchial infection and chronic bronchitis of childhood . It was The confirmed diagnosis of PBB depended on positive bacterial cultures of sputum or BALF . Most ofFor the pathogen analysis, the most common pathogens in our center were Spn 17.3%) and Sa (5.8%), and the top five pathogens were Spn, Hi, Mcat, Sa, and Pae in the published Chinese research. The data from the western countries revealed that the main pathogenic bacteria were Hi, Mcat, Spn, and Sa. The top three pathogens were the same in the east and west, and Spn and Hi were the most frequent etiology in China and the western countries, respectively. This result may be caused by the inconsistent distribution of pathogenic bacteria and the choice of antibiotics in different countries and regions. Co-infection could be found in the identified studies, some patients were caused by multiple bacteria and several cases were co-infected by bacteria and viruses 7.3% and . ConsistStudies showed that PBB occurred mainly in younger than 3 years old males . In ChinAmo was preferred for the treatment of PBB since it was sensitive to the common pathogens . StudiesStudies suggested that PBB, chronic suppurative lung and bronchiectasis were in a dynamic development process. PBB was considered to be the early stage of chronic suppurative lung disease and it shared some similarities with early bronchiectasis . PBB andThis study has the limitation that the sample size of our center was relatively small. The second limitation is the possibility of bias may exist because of the retrospective design. In addition, the follow-up was relatively short which may be difficult to reflect the outcome of PBB. Nevertheless, the combined analysis of the published data from the east and west limits this potential bias.In conclusion, PBB is common in male infants with chronic wet cough and may be accompanied by wheezing and airway deformities. Most cases are clinically diagnosed PBB in China and microbiologically-based-PBB is common in western countries. Co-infection could be found, and Spn, Hi, Mcat are the main pathogens. Spn and Hi were the most frequent etiology in China and western countries, respectively. Patients with comorbidities may need extended antibiotics treatment for more than 2 weeks.


Pressure injuries, also known as pressure ulcers, are local skin injuries. Once a pressure injury occurs, clinical treatment is relatively difficult, the treatment cycle is long, and the treatment cost is high, which brings heavy burdens to patients and society. Therefore, look for a reliable pressure injuries treatment method is 1 of the focus of clinical nursing workers.At present, there are many kinds of dressings to treat pressure injuries, and there is no uniform conclusion about which dressing is the most effective. Therefore, we systematically evaluate the effects of different dressings on the treatment of pressure injuries.We systematically searched the Chinese and English databases: PubMed, Embase, CENTRAL, CINAHL, Web of Science, CNKI, CBM, VIP, Wan Fang. Literature screening, data extraction, and quality evaluation were carried out by 2 researchers, and finally, use R software to carry out network meta-analysis.This study is ongoing and the results will be submitted to a peer-reviewed journal for publication.Ethical approval is not applicable, since this is an overview based on published articles.INPLASY2020100087. It is common in patients who have been bedridden for a long time. It is mainly caused by medical operations or compression by medical devices. Once PIs occurs, it is prone to secondary infection, which will affect the surgical prognosis of patients, lead to the prolonged hospital stay and increased medical costs, which not only increases the burden of treatment and care, but also reduces the quality of life of patients. It not only brings physical and psychological pain to patients, but also is a public economic health problem, which deserves the attention of the majority of medical and health personnel.,6 An epidemiological study conducted in Europe showed that the prevalence rate of PIs was 1.8% to 53.2%.–9 The medical expenses for treatment and care of PIs accounted for about 3.5% to 5.0% of the national public health expenditure.,11 Studies in the United States have shown that pressure ulcers can increase mortality, and deaths from stress injuries to instructors account for 0.4 percent of all deaths in the United States.Pressure injuries (PIs), also known as pressure ulcers, is a common and severe complication in clinical nursing work, and is also an important indicator to measure the quality of nursing. Therefore, finding a reliable treatment method for pressure ulcers is 1 of the priorities of clinical nursing workers. The current local treatment of PIs mainly uses various wound dressings to promote wound healing, help debride the wound, reduce bacterial load, and prevent further injury. In recent years, there have been more and more studies on dressings to treat PIs. However, at present, there are many kinds of dressings for the treatment of pressure injury, and each has its advantages, and there is no unanimous conclusion.PIs and its treatment are some of the most challenging clinical problems in hospitals.,15 A good NMA of the randomized controlled trial (RCTs) is considered the best quality evidence to provide sufficient information for practice.,17 It is also a significant source of critical information for researchers.,19 In the same type of research subjects, the NMA can systematically compare several different kinds of interventions for a particular problem, and rank them according to the effect of a specific outcome indicator, to obtain the best intervention plan. Based on this, this NMA evaluated the effects of different dressings in the treatment of pressure injuries, to provide evidence for the clinical selection.In the last decade, network meta-analysis (NMA) has been introduced.22.1https://inplasy.com/inplasy-2020-10-0087/).This NMA has been registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY). The registration number is INPLASY2020100087, DOI number is 10.37766/inplasy2020.10.0087(2.22.2.1Inclusion: RCTs were published in Chinese or English language without restriction on blind methods.Exclusion:(1)Non-Chinese and English literature;(2)Incomplete or missing research data;(3)Unable to obtain original documents;(4)Repeated publication of literature;(5)Editorials(6)Commentaries.2.2.2Patients of any age were described as having PIs. Studies were excluded if the study included other types of wounds (such as chronic wounds and venous leg ulcers) or if the subjects were animals.2.2.3Hydrocolloid dressings, silver dressings, foam dressings, saline gauze, petrolatum gauze, collagen dressings, danghui dressings, honey dressings, and other dressings or conventional treatment.2.2.4Main outcomes:(1)Effectiveness: Time to complete healing/rate of healing;(2)Safety: wound infection, bacteria amount, pain during treatment;(3)Cost.Additional outcomes: length of hospital stays, the incidence of different types of infection.2.32.3.1We will search the following English electronic bibliographic databases: PubMed (inception- present), Embase (inception- present), Cochrane Central Register of Controlled Trials (inception- present), CINAHL (inception- present), Web of Science (inception- present), as well as the Chinese databases: China Knowledge Network (inception- present), China Biomedical Literature Database (inception- present), VIP Data(inception- present), Wan Fang Data(inception- present).2.3.2Furthermore, reference lists of included RCTs and relevant systematic reviews will be searched. There will be no restrictions on publication year.2.3.3All databases will be based on the MeSH and text word search will be adjusted according to the specific database. Take PubMed as an example, and the searching strategy is shown in Table 2.3.4All search results are imported into ENDNOTE X8 literature management software; Two researchers  were conducted separately, and the relevant literature was screened strictly according to the research purpose and inclusion criteria, and the third researcher (YJW) was requested to judge if there were divergent literature.2.3.5After careful reading of the included literature, we will use Microsoft Excel 2013 to create a pre-determined data extraction table to collect relevant information and data, including general data , sample size, intervention measures, and outcome indicators, etc. The data will be extracted independently by 2 reviewers . Any differences will be settled through discussions between the 2 reviewers or by the third researcher (HLL).2.4The methodological quality of the final included RCT will be evaluated independently by 2 reviewers . Any disagreements will be resolved through discussion between the 2 parties or decided by a third reviewer (YJW).Evaluate the quality of the literature according to the recommended bias risk assessment tool Cochrane 5.1.0. The evaluation contents include: random sequence generation, allocation concealment, blind method of participants, researchers and result evaluators, the integrity of outcome indicators, selective reporting, other source bias, and so on. Each was rated as “high risk of bias,” “unclear,” and “low risk of bias.”2.52.5.1I2 statistic and P value. I2 was used to judge the size of heterogeneity, I2 ≤50%, it can be considered that the homogeneity among studies is good; If I2 > 50%, it is considered that the heterogeneity among studies is large, and multiple regression model is adopted for processing.R 3.5.0 Software gemtc package and JAGS 3.4.0 software were used for data analysis, and Stata 15.0 was used to draw the network diagram and funnel diagram. We will calculate the mean differences or standardized mean differences with 95% confidence interval for continuous variable data, and relative risk with 95% confidence intervals for dichotomous variable data. Set the number of pre-iterations to 10,000 and the number of iteration operations to 100,000. The statistical heterogeneity will be examined using the 2.5.2P < .05, the direct comparison results were inconsistent with the indirect comparison results. If P ≥.1, I2 < 50%, it indicates that there is homogeneity among the studies or the heterogeneity is within the acceptable range, and the fixed effects model is used to merge the calculation of the effect size; on the contrary, it is considered that there is heterogeneity between the studies. Egger method and Begg method were used to assess publication bias.The consistency test was judged by the node-splitting model. When 2.5.3If the evidence is sufficient, we will conduct a subgroup analysis to determine the difference between different gender, age  and so on.2.6Two reviewers (YYC and YJW) will use the Grading of Recommendations Assessment, Development and Evaluation method to assess the quality of evidence of included studies. The evidence levels are classified into 4 levels: high, moderate, low, or very low.2.7A “summary of findings” table will be created for the main outcomes, with hydrocolloidal dressing and saline gauze as intervention measures, and the outcome index is mainly the proportion of ulcers completely healed, wound infection, pain during treatment. Finally, we will complete the table after analyzing all the article data, and refer to Table 3We identified 5243 records through database searching and 4 records through other sources. The detailed search flowchart is shown in Figure 3.1–26We conducted preliminary experiments and included 7 RCTs. Of the 7 RCTs, 2 were from China, 2 were from the United States, 1 was from Iran, and 2 were not mentioned in the article. The total sample size is 299, among the interventions, saline gauze and hydrocolloid dressings are the most, and the subjects are basically pressure ulcer patients with stage 2 and above. For further details, please refer to the characteristics of some of the included studies Table .[20–26]4Dressings are widely used in wound care, with the aim of protecting the wound and promoting healing. However, there is no consensus on the efficacy, safety and health economy assessment of dressings. So, we did this network meta-analysis to analyze the different dressings and provide a reference for clinical practice.Conceptualization: Yitong Cai, Yuying Zhou.Methodology: Yitong Cai, Lina Xing, Yingying Kang.Software: Yitong Cai, Hailing Li.Writing – original draft: Yitong Cai, Peng ChengWriting – review & editing: Yitong Cai, Yujuan Wang.


The coronavirus disease (COVID-19) has been identified as the cause of an outbreak of respiratory illness in Wuhan, Hubei Province, China beginning in December 2019. As of 31 January 2020, this epidemic had spread to 19 countries with 11 791 confirmed cases, including 213 deaths. The World Health Organization has declared it a Public Health Emergency of International Concern.A scoping review was conducted following the methodological framework suggested by Arksey and O’Malley. In this scoping review, 65 research articles published before 31 January 2020 were analyzed and discussed to better understand the epidemiology, causes, clinical diagnosis, prevention and control of this virus. The research domains, dates of publication, journal language, authors’ affiliations, and methodological characteristics were included in the analysis. All the findings and statements in this review regarding the outbreak are based on published information as listed in the references.Most of the publications were written using the English language (89.2%). The largest proportion of published articles were related to causes (38.5%) and a majority (67.7%) were published by Chinese scholars. Research articles initially focused on causes, but over time there was an increase of the articles related to prevention and control. Studies thus far have shown that the virus’ origination is in connection to a seafood market in Wuhan, but specific animal associations have not been confirmed. Reported symptoms include fever, cough, fatigue, pneumonia, headache, diarrhea, hemoptysis, and dyspnea. Preventive measures such as masks, hand hygiene practices, avoidance of public contact, case detection, contact tracing, and quarantines have been discussed as ways to reduce transmission. To date, no specific antiviral treatment has proven effective; hence, infected people primarily rely on symptomatic treatment and supportive care.There has been a rapid surge in research in response to the outbreak of COVID-19. During this early period, published research primarily explored the epidemiology, causes, clinical manifestation and diagnosis, as well as prevention and control of the novel coronavirus. Although these studies are relevant to control the current public emergency, more high-quality research is needed to provide valid and reliable ways to manage this kind of public health emergency in both the short- and long-term. The coronavirus belongs to a family of viruses that may cause various symptoms such as pneumonia, fever, breathing difficulty, and lung infection . These vIn response to the outbreak, the Chinese Center for Disease Control and Prevention (China CDC) dispatched a rapid response team to accompany health authorities of Hubei province and Wuhan city to conduct epidemiological and etiological investigations. The WHO confirmed that the outbreak of the coronavirus epidemic was associated with the Huanan South China Seafood Marketplace, but no specific animal association was identified . ScientiGiven the spread of the new coronavirus and its impacts on human health, the research community has responded rapidly to the new virus and many preliminary research articles have already been published about this epidemic . These online databases contain archives of most English and Chinese biomedical journals. In addition, some white papers published online by the National Health Commission of the People’s Republic of China, Chinese Center for Disease Prevention and Control, and the WHO were included in the analysis. We searched scientific publications from 1 January to 31 January 2020. The search terms were ‘nCoV’, ‘2019 novel coronavirus’, ‘2019-nCoV’, ‘novel coronavirus’, ‘Pneumonia’, ‘新型冠状病毒’ (Chinese), ‘新型肺炎’ (Chinese), and ‘新冠病毒’ (Chinese). We included all the relevant scientific publications written in English or Chinese in the review. Non-scientific commentary, reports, and news articles were excluded from the analysis.Two researchers (YW and SPA) independently searched through the literature. The two sets of literature were then compared. Disagreements on the inclusion or exclusion of literature were resolved through discussion or, if necessary, by including a third researcher (HZ) to make the final decision. Duplicate articles were eliminated. Eventually, 65 unique academic publications were included in this analysis ; ‘causes’ includes studies on virology, pathogenesis, and transmission patterns; ‘clinical manifestation and diagnosis’ includes studies on the clinical features (signs and symptoms) and diagnosis; and ‘prevention and control’ includes studies on prevention, control, and treatment measures.Authors’ affiliations were used to categorize the type of research collaboration. A publication was considered as deriving from ‘inside China’ when all affiliations of the authors were from China; a publication was considered as deriving from ‘outside China’ when all affiliations were outside China; a publication was considered as deriving from ‘international collaboration’ when at least one affiliation was from China and at least one was from outside China. The research domains, publishing dates, journal language, authors’ affiliations, as well as methodological characteristics were analyzed respectively. All findings and statements regarding the outbreak in this review are based on published information as listed in the references.The Lancet and The New England Journal of Medicine. Most of the publications  are in English and few  are in Chinese. Around 75.9% of English language publications focus on epidemiology and causes, while 85.7% of Chinese papers focus on prevention and control  are related to causes, followed by papers on epidemiology , while 18.5% examined prevention and control and 13.8% reported clinical manifestations and diagnosis. Initially, there were more research articles focused on causes of the outbreak, yet studies on prevention and control gradually increased over time  were published by Chinese scholars whereas 29.2% (n = 19) articles were from scholars outside of China. A small number  were based on international collaborative research by scholars from different countries  and cross-sectional study designs (18.5%). Around half of the studies include targeted populations  (49.2%) in their analysis, followed by studies that attempt to identify the species of the virus  36.9%). Among the 32 articles with population as the target of study, 34.4% had a sample size of less than 10, 31.3% were conducted in a hospital setting, and 50% used secondary data. Quality control activities for data collection were mentioned in 56.3% of the population studies  . It appe0) range to be from 2.6 to 4.71  of COVID-19 2.9) is estimated to be higher than the reported effective reproduction number (R) of SARS (1.77) at this early stage . Differe.9 is estIn China, 11 791 cases were confirmed and 17 988 cases were suspected in 34 provinces as of 24:00, 31 January 2020 Fig.  33].Fi.Fi33].Coronaviruses are enveloped single-stranded RNA viruses that are zoonotic in nature and cause symptoms ranging from those similar to the common cold to more severe respiratory, enteric, hepatic, and neurological symptoms , 34. OthTo detect the infection source of COVID-19, China CDC researchers collected 585 environmental samples from the Huanan Seafood Market in Wuhan, Hubei Province, China on 1 January and 12 January 2020. They detected 33 samples containing SARS-CoV-2 and indicated that it originated from wild animals sold in the market . Then, rTo conduct next-generation sequencing from bronchoalveolar lavage fluid and cultured isolates, researchers enrolled nine inpatients in Wuhan with viral pneumonia and negative in common respiratory pathogens. The results of this next-generation sequencing indicated that SARS-CoV-2 was more distant from SARS-CoV (with about 79% sequence identity) and MERS-CoV (with about 50% sequence identity) than from two bat-derived SARS-like coronaviruses – bat-SL-CoVZC45 (with 87.9% sequence identity) and bat-SL-CoVZXC21 (with 87.2% sequence identity) . StudiesMany domestic and wild animals, including camels, cattle, cats, and bats, may serve as hosts for coronaviruses . It is cThe latest guidelines from Chinese health authorities , 43 descThe complete clinical manifestation is not clear yet, as the reported symptoms range from mild to severe, with some cases even resulting in death . The mosFor patients with suspected infection, the following procedures have been suggested for diagnosis: performing real-time fluorescence (RT-PCR) to detect the positive nucleic acid of SARS-CoV-2 in sputum, throat swabs, and secretions of the lower respiratory tract samples , 14, 43.Prevention and control strategies and methods are reported at three levels: national level, case-related population level, and general population level. At the national level, the National Health Commission of the People’s Republic of China issued the “No.1 announcement” on 20 January 2020, which officially included the COVID-19 into the management of class B legal infectious diseases, and allowed for class A infectious disease preventive and control measures to be implemented . Under tTo date, no specific antiviral treatment has been confirmed to be effective against COVID-19. Regarding patients infected with COVID-19, it has been recommended to apply appropriate symptomatic treatment and supportive care , 16. TheFor the general population, at this moment there is no vaccine preventing COVID-19. The best prevention is to avoid being exposed to the virus . AirbornThe WHO also issued detailed guidelines on the use of face masks in the community, during care at home, and in the health care settings of COVID-19 . In thisIn addition to articles published in research journals, the China CDC published a guideline to raise awareness of the prevention and control of COVID-19 among the general population. The key messages of the guideline include causes, how to choose and wear face masks, proper hand washing habits, preventive measures at different locations , disinfection methods, and medical observation at home . In addiThe review applied a systematic and rigorous search strategy to retrieve relevant articles according to the research objectives. This research summarizes scientific foundations, identifies literature gaps, and suggests some evidence for future research directions on COVID-19 which will provide information for research community, policymakers, and health professionals to adjust and/or come up with new research, policies, and practices. Our study only focuses on the articles published either in English or in Chinese during the early outbreak period. Although, it cannot reflect the entire body of research on COVID-19 worldwide, it will provide some evidences for future study and control.This study shows a holistic picture of the current research in response to the outbreak of COVID-19. During this early period, many studies have been published exploring the epidemiology, causes, clinical manifestation and diagnosis, and prevention and control of the novel coronavirus. Thus far, most studies have focused on the epidemiology and potential causes. However, studies exploring prevention and control measures have begun to gradually increase. Studies in this domain are urgently needed to minimize the impact of the outbreak. Government agencies have quickly incorporated recent scientific findings into public policies at the community, regional, and national levels to slow down and/or prevent the further spread of the COVID-19. We recommend that the scholarly community conduct further research to provide valid and reliable ways to manage this kind of public health emergency in both the short-term and long-term.Additional file 1. Published research articles on COVID-19 in January 2020.Additional file 2. Clinical trials registered as of 31 January 2020.


Lactobacillus in silage after aerobic exposure. The enrichment of L. buchneri was significant in ML silage at days 5 and 7 during the aerobic stage. The abundance of harmful microorganisms, such as aerobic bacterial including Acinetobacter, Providencia, Bacillus, and yeasts including Issatchenkia, Candida, and Kazachstania, were suppressed in ML silage. M and L had an impact on bacterial and fungal microbes, resulting in the improvement of fermentation quality and reduction of aerobic spoilage in barley silage.This study aimed to investigate the effects of microbial inoculants (L) and molasses (M) on the bacterial and fungal microbiomes of barley silage after the aerobic stage. The addition of molasses and microbial inoculants improved the aerobic stability of barley silage. The ML silage, which had a low pH value and high lactic and acetic acid contents, remained aerobically stable for more than 216 h. The ML silage exhibited low bacterial and high fungal diversities. Microbial inoculants and molasses enriched the abundance of  Selective additives, such as lactic acid bacteria (LAB), are used to reduce aerobic spoilage and the accumulation of toxic matter in the progress of ensiling2. In our previous study, we investigated the effects of LAB on bacterial and fungal microbiomes during aerobic exposure and found that LAB inoculants improved the aerobic stability of barley silage, but the pH value was 4.85 after 7 d of aerobic exposure. Issatchenkia was the main microorganisms that caused aerobic spoilage in barley silage; however, the addition of LAB has little effect on fungal communities3. The use of silage additives individually or in combination during ensiling should reduce decomposition and prolong the stability of barley silage upon air exposure.The growth and reproduction of aerobic bacteria (AB), yeasts, and molds usually lead to the poor fermentation quality of silage upon aerobic exposure4. Studies have reported that the addition of M results in reducing pH values and increasing lactic acid (LA) concentrations in silages6. Several studies have focused on the effects of additives or M on fermentation characteristics and aerobic stability7, whereas others have examined silage quality and the dynamics of microbial community8. The addition of molasses had no pronounced effects on fermentation quality of alfalfa silage, but increased aerobic stability9. The simultaneous application of L. plantarum inoculant and molasses improved fermentability and aerobic stability of sawdust-based spent mushroom substrate silages10. However, few studies have comprehensively evaluated fermentation quality, aerobic stability, and microbial community dynamics in barley silage prepared with M and LAB after aerobic exposure, which may provide important data regarding aerobic stability of barley silage.Molasses (M) is used as a fermentation stimulant to increase the rate of silage acidification by providing fermentable sugars for the growth of LAB during the ensiling of silage11. Moreover, the composition and shift of microorganisms, including bacteria and fungi, determines fermentation quality and aerobic stability of barley silage. Hence, the identification of microbial shifts in barley silage with the addition of M and LAB is essential.Microbial communities are essential to conserve barley silage. Next-generation sequencing (NGS) is an approach for the characterization and composition of microbial communities of silageTherefore, NGS high-throughput sequencing was used in this study to analyze silages characteristics and bacterial and fungal microbiomes of barley silage with the addition of M and LAB during aerobic stage.p < 0.05). LA and AA contents were higher in the ML silage than the CK and M silages at days 2, 5, and 7 of aerobic exposure (p < 0.05). In addition, as shown in Fig. p < 0.05). Nevertheless, the yeast and AB counts of the ML group were significantly lower than those of the CK and M groups during aerobic exposure (p < 0.05).The pH values of the control (CK), M silage and ML silage were within the range of 3.87–3.94 after 60 days of ensiling Table . When exIn this study, the operational taxonomic units and Chao 1 indices, which represented the richness of bacterial communities, had notably decreased in barley silage treated with ML at 2 and 5 d during aerobic stage Table . FurtherAmong the three groups, the operational taxonomic units, Chao 1, and Shannon indices of fungi were the highest in ML silage after 7d of exposure. Nevertheless, these indices decreased in the ML silage during aerobic stage. These findings show that the richness and diversity of fungal microbes had increased in barley silage treated with ML after aerobic exposure.Firmicutes was the most abundant phyla with more than 97% relative abundance in ML silage during aerobic stage  of effect size (LEfSe), which was used to calculate the relative abundances of bacterial genera in barley silage at 5 and 7 d during aerobic stage Fig. . LEfSe wLactobacillus, Weissella, and Pediococcus were positively correlated with LA, AA, and PA concentrations, and LAB counts but negatively correlated with pH values and AB. Acinetobacter was positively correlated with pH values, as well as yeast and AB counts, but negatively correlated with LA concentrations and LAB counts. Stenotrophomonas and Empedobacter were positively correlated with pH values, as well as yeast and AB counts, but negatively correlated with LA and PA concentrations, and LAB counts.The results of correlation analysis of bacterial communities, silage fermentation, and microbial counts of barley silage during aerobic stage are shown in Fig. Figure Ascomycota phylum were more abundant in M and CK silages with an abundance of more than 97% during the aerobic stage. Basidiomycota in the ML silage was the preponderant fungi at the early stage of exposure, with abundances of 62.562% and 78.714% at 0 and 2 d, respectively, during the aerobic stage. Ascomycota was the most abundant group in the ML silage at 5 d of aerobic exposure. Figure Issatchenkia, Kazachstania, and Candida with abundances of 80.580%, 12.055%, and 4.449% respectively. Meanwhile, Issatchenkia, Monascus, and Kazachstania were the major fungi in M treated silage at 2 d during aerobic stage with the abundances of 55.310%, 35.024%, and 5.487%, respectively. Compared with CK silage, the addition of M decreased the abundance of Issatchenkia and increased the abundance of Monascus and Candida at 0 and 2 d during aerobic stage. Furthermore, the abundances of Issatchenkia, Candida, and Kazachstania decreased in the ML silage after aerobic exposure, as compared with those of the CK and M silages. However, other genera (less than 1% sequenced) were the predominant fungi in ML silage at 2 d during aerobic stage at 82.5% abundance, followed by Issatchenkia (8.95%). This result was in accordance with fungal diversity of barley silage, which showed that ML silage had the greatest fungal diversity at 2 d during aerobic stage among the three groups, as shown in Table Sporidiobolus was a indicator of aerobic stability in the ML silage at 0 d during aerobic stage, although the abundance of Kazachstania in CK silage was significantly enriched at 2 d during aerobic stage. The relationship between the fungal community structure and fermentation quality , decreased the pH value and content of yeast, and increased the contents LA and LAB of barley silage after aerobic exposure.Fungal community composition at the phylum level in ML silage after aerobic stage was significantly different with CK silage Fig. . The mem13. Consistently, in this study, the pH values of barley silage prepared with ML were low, suggesting ML silage has high aerobic stability. During aerobic exposure, the pH values were always less than 4.0 in ML silage, while the pH value reached 4.85 in barley silage added with inoculants at day 7 of aerobic exposure3. The yeast and AB counts of the ML group were significantly lower than those of the CK and M groups during aerobic exposure. Meanwhile, the counts of yeast and AB in ML silage were also less than those of barley silage added with inoculants which reported by Liu et al.3 at day 5 and 7 of aerobic exposure. Yeasts, as primary initiators of aerobic spoilage in silage, assimilate lactate and degrade it into CO2 and water, which is accompanied with nutrient loss in silage exposed to air. As a result of lactate depletion by yeasts, pH increases, rendering silage more susceptible to spoilage14. Compare to LA, it is easier for yeasts to be inhibited by AA15. According to the report by Danner et al.16, silages with high concentrations of AA have low populations of yeasts and molds and high aerobic stability. Barley silage treated with ML had the highest AA content during the aerobic stage in this study. The lowest yeast population was observed at 7 d of aerobic exposure, indicating that the aerobic stability of ML silage was the highest among the groups. However, these results do not conform to those of Kim et al.10, who reported that silages treated with M and L. plantarum had the lowest AA content, the highest populations of yeasts, and the highest aerobic stabilities during the ensiling of silage.The pH value was one of the main factors that influenced the extent of fermentation and quality of silage. A low pH value of 3.94 or less at the terminal point of silage indicates good fermentation during ensiling and aerobic exposure. The decline in microbial growth, which may favor the growth of undesirable anaerobic organisms and subsequently decrease aerobic stability, was effectively inhibited in silage under low pH conditionsL. plantarum. This result is supported by the findings of a previous study conducted by Méndez-García et al.17, who indicated that limited microbial diversity was due to low pH values in acidic environments. A similar result was reported by Yang et al.18. Lower bacterial diversity and greater aerobic stability were also observed in silages with L. buchneri and PA than those of CK silages19. However, bacterial diversity did not decrease in M silage, as compared with CK silage, but was evidently reduced in ML silage during aerobic exposure in our study. Together, these results show that the richness and diversity of microbial communities in silage were affected by the addition of ML during aerobic exposure.After aerobic exposure, the diversity and richness of bacteria in ML silage were lower than that in CK and M silages, which might be attributed to the predominant roles of Firmicutes are gram-positive bacteria with genomes having low G + C contents and most degrade macromolecular compounds, such as cellulose, protein, and starch20. McGarvey et al. reported that Firmicutes and Proteobacteria were the dominant bacteria in alfalfa silage21. Proteobacteria is the largest phylum of bacteria and includes pathogenic bacteria, such as Escherichia coli, as well as Vibrio and Helicobacter spp., in addition to Erwinia, Sphingomonas, Methylobacter, Pseudomonas, and Agrobacterium spp.22. Romero et al. indicated a relatively high abundance of Firmicutes (99.8%) and a low abundance for Proteobacteria (0.07%) in wilted whole-crop oats ensiled for 217 days20. In this experiment, the bacterial community structure in silages changed substantially with prolonged exposure. Similar to the findings of Peng et al.22, in this study, the abundance of Firmicutes was significantly decreased, whereas that of Proteobacteria increased at 7 d during the aerobic stage in the CK and M silages. During aerobic exposure, the dominant phylum in ML silage was Firmicutes, with the abundance of >97%, which was significantly higher than that of barley silage added with inoculants. According to the findings of Liu et al.3, the dominant phylum in barley silage added with inoculants was Firmicutes, with the abundance of >99% at day 0 and 2 of aerobic exposure. However, Firmicutes and Proteobacteria were dominant in barley silage added with inoculants, with the abundance of 78.9% and 23.0% at day 5 of aerobic exposure, 20.8% and 69.2% at day 7 of aerobic exposure, respectively. According to a report by Xu et al.23, the abundance of Firmicutes predominated in spoiled silage after aerobic exposure, but was subsequently replaced by Proteobacteria and Actinobacteria.Lactobacillus prevented aerobic spoilage due to the production of LA and the reduction of pH value24. In this study, the abundance of Lactobacillus predominated in ML silage throughout aerobic exposure, reaching more than 95%. At day 0 and 2 of aerobic exposure, Lactobacillus was dominant in CK silage and M silage, with the abundance of >94%. However, the abundance of Lactobacillus in M silage was only 48.2% and 1.2% at day 5 and 7 of aerobic exposure, respectively. Liu et al.3 reported that the abundance of lactobacillus for dominant genera in barley silage with inoculants kept more than 99% at day 0 and day 2 of aerobic exposure, but the abundance decreased to 71.4% and 11.6% at day 5 and 7 of aerobic exposure, respectively. Therefore, the addition of molasses combined with microbial inoculants could improve the structure of bacteria community which was predominated by Lactobacillus in barley silage during aerobic exposure. Pang et al.25 found that Lactobacillus was the prominent genus in sorghum, forage paddy rice, and alfalfa silages. M provides additional fermentable substrates for LAB, which promotes domination in the microbial communities of silage, thereby directing the metabolism to homofermentative LAB26. Li et al.27 reported that AA and PA might exert direct effects on bacterial physiology and indirect effects on the microbial community structure. In this study, the AA and PA concentrations in the ML group might be sufficiently high to effectively inhibit the growth of aerobic microorganisms that cause spoilage. With the addition of ML during the ensiling of barley silage, Lactobacillus, as beneficial bacteria, became the absolute dominant genus and competitively inhibited the growth of harmful bacteria, such as Empedobacter and Acinetobacter. The low bacterial diversity observed in ML silage, as indicated by the low Shannon index of 1.518, was a result of the high abundance of Lactobacillus species at 95.458% and low pH value (3.98) of barely silage after 7 d of exposure. Lactobacillus buchneri is a heterofermentative species that produces a variety of products and ferments LA into AA29. The conversion of LA to AA results in high concentrations of AA, which is regarded as an inhibitor of yeasts and molds. In this study, at 5 and 7 d during aerobic stage, LA and AA concentrations were higher in the ML silage than the CK and M silages. Hence, barley silage ensiling with ML had the best aerobic stability among the three groups. This result is possibly due to L. buchneri from ML silage, which plays an important role in pH, fermentation products, and microbial community dynamics during aerobic exposure.et al.30 deduced that the proliferation of bacilli normally occurs in the later period of aerobic spoilage, as indicated by the detection of Bacillus spores at the outermost layers of grass and maize silages. According to Liu et al.31, Stenotrophomonas possibly caused aerobic deterioration of corn straw silage. Acinetobacter are aerobic non-fermenting bacteria that exist in various environments except silage32. According to Liu et al.31, AB in corn straw silage upon aerobic exposure mainly comprises Acinetobacter, Stenotrophomonas, and Bacillus, which possibly cause aerobic deterioration of corn straw silage. In the present study, these aerobic bacteria were positively correlated with pH values, as well as yeast and AB counts, and promoted the growth of spoilage microorganisms.Graf et al.33, who reported that silage treated with L. buchneri, which was stable after aerobic exposure, differed from untreated silage at day 90 in terms of three pathways; namely, base-excision repair, pyruvate metabolism, and transcription machinery. Notably, a high degree of pyruvate metabolism was also observed in LAB-supplement barley silage, suggesting a possible reason for the enhancement of silage stability3. Amino acids were metabolized by the dominant microflora in L. plantarum supplement and untreated silages during the ensiling33. The addition of L. buchneri increased the accumulation of polyols and free amino acids, which was possibly due to the unique metabolic pathways of sugar fermentation and amino acid biosynthesis executed by L. buchneri34. In the present study, the changes in the carbohydrate and amino acid metabolic pathways may have contributed to the inhibition of aerobic spoilage in ML silage. As compared with the CK silage, the higher abundances of carbohydrates and amino acids were responsible for the improved aerobic stability of LAB-inoculated barley silages3.As compared with the ML group, the microbiota in the CK group had higher functional performance related to bacterial secretion systems, bacterial motility proteins, two-component systems, and adenosine triphosphate binding cassette transporters, which are involved in environmental information processing. This result is in agreement with those of Keshri Ascomycota phylum were more abundant in M and CK silages with an abundance of more than 97% during the aerobic stage. These results were similar to the findings of Romero et al.20, who reported that Ascomycota was the most abundant group, and Basidiomycota was the second most abundant in oat silage. In our study, Basidiomycota in the ML silage was the preponderant fungi at the early stage of exposure during the aerobic stage. Ascomycota was the most abundant group in the ML silage at 5 d of aerobic exposure, which was accompanied with a decrease in the abundance of Basidiomycota, suggesting that the fungal community structure was significantly modified by ML treatment at the early stage of aerobic exposure. Yeasts, such as Issatchenkia, Candida, and Saccharomyces spp, are considered basic initiators of aerobic spoilage in barley silage35. The abundances of Issatchenkia, Candida, and Kazachstania decreased in the ML silage after aerobic exposure, as compared with those of the CK and M silages, suggesting the addition of ML affected fungal community composition. Issatchenkia was the main microorganism causing aerobic deterioration of corn and barley silages36. Liu et al.3, indicated the addition of inoculants could decrease the abundances of Issatchenkia at day 0 of aerobic exposure, but not affect the abundances of Issatchenkia at day 2, 5 and 7 of aerobic exposure. Our results suggested the addition of molasses combined with microbial inoculants could affect fungal community composition and decrease the abundances of Issatchenkia in barley silage, however, single addition of inoculants had little effect on fungal community composition during aerobic exposure. The abundance of harmful microorganisms, such as aerobic bacteria including Acinetobacter, Providencia, Bacillus, and yeasts including Issatchenkia, Candida, and Kazachstania, were inhibited in the ML silage. Sporidiobolus can be exploited for biotechnological production of β-carotene using conventional fed-batch fermentation37. Sporidiobolus and Sporobolomyces produce carotenoids, which are effective antioxidants having potential antimicrobial properties38. In addition, similar results reported by Dolci et al. showed that the dominant yeast species in corn silage was Kazachstania exigua after exposure to air, as determined by denaturing gradient gel electrophoresis profiles of fungal DNA and RNA39. According to Lu et al., Kazachstania unispora was cardinal among the dominant species of yeasts in aerobically deteriorating corn silage40.The members of Lactobacillus was enriched, while that of harmful microorganisms, such as aerobic bacterial including Acinetobacter, Providencia, Bacillus, and yeasts including Issatchenkia, Candida, and Kazachstania, were lowest in the ML silage. The addition of ML could increase AA content, reduce yeast and AB counts, change bacterial and fungal microbes, improve fermentation quality, and reduce aerobic spoilage of barley silage.ML silages remained aerobically stable for more than 216 h with stable pH values and LA contents. Bacterial diversity was reduced, while fungal diversity was increased in the ML silage during aerobic exposure. The abundance of L. plantarum, L. casei, and L. buchneri (2:1:1)3, which were isolated form ryegrass silage, rice straw silage, corn silage, respectively. The NCBI accession number of inoculants were MK106013, MK106014, MK106015, respectively. The combined inoculants were added at 5 × 105 colony forming units (CFU) g−1 of fresh material (FM). The chopped barley was mixed and divided into equal portions for three treatments comprising no additive (CK group), 2 g of M per 100 g of FM (M group), or 2 g of M per 100 g of FM + inoculants (ML group). After thorough mixing, barley was ensiled in 1 L silos.Barley was harvested from a farm located in Yancheng, Jiangsu, China  in May 2017. Barley was chopped using fully automatic harvester. We used inoculants and molasses as additives for barley silage. The inoculants were prepared by our laboratory and comprised 41.After 60 days of ensiling, silos were opened. Fermentation quality, microbial counts, chemical compositions, bacterial and fungal community structures were analyzed at 0, 2, 5, and 7 d during aerobic exposure. The aerobic stability was determined using a previously described method2SO4; flow rate, 0.5 mL/min). Approximately 100 g of each sample was dried at 55 °C for 48 h and weighed to determine the dry matter content of barley silage. Both neutral and acid detergent fibers were analyzed using the method described by Van Soest et al.42.The pH value and organic acid concentrations were determined by homogenizing 20 g of silage in 180 mL of sterilized water at 4 °C for 24 h and then filtrating the homogenate through four layers of cheesecloth. The pH of the filtrate was measured using a glass electrode pH meter . LA, acetic acid (AA), and propionic acid (PA) and ethanol were determined by high performance liquid chromatography . The analytical conditions were as follows: column, Carbomix® H-NP5 ; oven temperature, 50 °C; mobile phase, 2.5-mM Het al.3. The culture medium used were lactobacilli de Man, Rogosa, Sharpe agar , potato dextrose agar , and nutrient agar . Lactic acid bacteria were incubated at 30 °C for 48 h under anaerobic conditions, whereas yeasts and aerobic bacteria were incubated at 30 °C for 24 h. Colonies were counted in CFU/g FM−1 as viable numbers of microorganisms.Twenty grams of each sample in 180 mL of physiological saline was blended and serially diluted. Microbial counts of LAB, yeasts, and aerobic bacteria (AB) were measured according to the study of Liu et al. and Zhang et al.43. The raw reads were deposited in the European Nucleotide Archive (ENA) database under the accession number ERP118410 and ERP118523.Total DNA was extracted using the PowerSoil® DNA Isolation Kit  in accordance with the manufacturer’s protocol. The V3-V4 region of bacterial 16S rDNA and the fungal internal transcribed spacer 1 region were amplified, sequenced, and analyzed according to the methods described by Liu p) value of <0.05 was considered statistically significant.Fermentation quality, microbial counts, nutritional value were analyzed by two-way analysis of variance for a 3 × 4 (treatment × storage period)-factorial arrangement of treatments and compared with Tukey’s test using IBM SPSS Statistics for Windows, version 19.0. . A probability (