The presented research aims to verify whether physicochemical properties of lung fibroblasts, modified by substrate stiffness, can be used to discriminate between normal and fibrotic cells from idiopathic pulmonary fibrosis (IPF). The impact of polydimethylsiloxane (PDMS) substrate stiffness on the physicochemical properties of normal (LL24) and IPF-derived lung fibroblasts (LL97A) was examined in detail. The growth and elasticity of cells were assessed using fluorescence microscopy and atomic force microscopy working in force spectroscopy mode, respectively. The number of fibroblasts, as well as their shape and the arrangement, strongly depends on the mechanical properties of the substrate. Moreover, normal fibroblasts remain more rigid as compared to their fibrotic counterparts, which may indicate the impairments of IPF-derived fibroblasts induced by the fibrosis process. The chemical properties of normal and IPF-derived lung fibroblasts inspected using time-of-flight secondary ion mass spectrometry, and analyzed complexly with principal component analysis (PCA), show a significant difference in the distribution of cholesterol and phospholipids. Based on the observed distinctions between healthy and fibrotic cells, the mechanical properties of cells may serve as prospective diagnostic biomarkers enabling fast and reliable identification of idiopathic pulmonary fibrosis (IPF). Interstitial lung diseases (ILD) form a heterogeneous group of disorders with varying degrees of inflammation and fibrosis, affecting mainly the interstitium of the lungs  ,2. ThougNowadays, the diagnostic process is based on a multidisciplinary approach involving pulmonologist, radiologist and pathologist experts and sophisticated examination techniques, such as high-resolution computed tomography (HRCT), and in some cases lung biopsy ,8,9,10, Making the proper diagnosis of IPF is hard. There are almost 200 different ILDs, and in most cases, their clinical manifestations are very similar if not the same. Additionally, there are a lot of similarities and overlap of the radiologic and histopathological patterns, which makes diagnosis even more difficult. In the year 2018, a multidisciplinary committee of IPF experts from the American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin American Thoracic Society provided ‘An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline’ formulating recommendations related to the diagnosis of IPF . HoweverIn IPF, the changes in cell properties, together with an extensive deposition of collagen and other components of the extracellular matrix (ECM), disrupt normal lung architecture and functions. The progression of fibrosis and inflammatory processes may lead to the development of dyspnea, cough, and ultimately, in some cases, respiratory failure and death . AlthougA lot of effort is made in the development of the prognostic indicators of IPF ,23. The In our previous work , we haveSylgard 184 (Dow Corning) was used to prepare PDMS substrates. First, the elastomer base was mixed with the curing agent (mass ratio of 10:1). Then, to enable the adjusting of substrate elasticity, a solution of benzophenone  in xylene  was added to the mixture, at the mass ratio of 1:100 benzophenone:PDMS and degassed. The PDMS substrates were fabricated using a spin-coating technique  on a 25 mm round coverslip glass. To produce soft substrates, some samples were additionally irradiated for 5 h with UV light (λ = 254 nm 400 W mercury lamp). In the last step, substrates were baked for 15 min at 150 °C .2 incubator providing 95% air/5% CO2 atmosphere. The PDMS substrates which were attached to glass coverslips were placed into the bottom of the Petri dish (35 mm in diameter) and they were sterilized for one hour under UVC light  under a laminar flow chamber . After that, cells  were placed over the all type of PDMS surface. Next, the Petri dishes were incubated in the CO2 incubator by 1, 3, or 6 days. For each experimental sequence, two or three identical samples were prepared and measured. All experiments were repeated at least three times for each cell line and a time-point to proving the reproducibility of the results.Human lung fibroblasts  were purchased from ATCC . Cells were cultured in the F-12K medium , which was supplemented with a 15% fetal bovine serum  and 1% antibiotic  in culture flasks, in a COCommercially available AFM system  equipped with two heads CellHesion or Nanowizard 4 was used to conduct force spectroscopy and quantitative imaging (QI), respectively. The PDMS substrates with cells were mounted in the AFM liquid cell filled with a culture medium (F-12K medium). The commercially available silicon nitride AFM probe  was immersed into medium and brought close to the surface. Force curves  were collected within a grid of 4 × 4 points, forming a 5 µm × 5 µm elasticity map. Each map was recorded for only one cell over the cellular nuclear region. In total, at least 40 cells were measured for each substrate and time point. To obtain the value of the relative Young’s modulus, an approach part of a recorded force curve was analyzed. It was converted into a force versus indentation curve to which the Hertz contact model was fitted . The shaLocal elasticity maps of normal and IPF-derived fibroblasts were recorded using the quantitative imaging (QI) capability of the Nanowizard 4 AFM head. These maps were recorded within a scan area of 50 µm × 50 µm (256 × 256 pixels).3 ion clusters generated by 30 keV bismuth liquid metal ion gun were used as primary ions. The ion dose density was kept below 1012 ion/cm2 to ensure static mode conditions and a pulsed low-energy electron flood gun was used for charge compensation.Imaging and molecular characterization of normal and IPF-derived fibroblasts were performed using TOF-SIMS 5 apparatus . BiFirstly, high resolution mass spectra (m/Δm > 7000) were acquired from at least 4 randomly chosen, non-overlapping spots (150 μm × 150 μm area). Next, collected mass spectra were analyzed using principal component analysis (PCA), to find alterations in surface chemical properties among the studied LL24 and LL97A cell lines. PCA was conducted using the PLS Toolbox  for MATLAB . Prior to PCA, the intensities of selected peaks from each spectrum were normalized to the sum of selected peaks and mean-centered. Finally, high-resolution images of both cell lines were collected in imagine mode from several non-overlapping 300 μm × 300 μm areas (with a resolution of 256 × 256 points).2 atmosphere, in the incubator (Nuaire) for 1 h. The MTS method is based on the reduction of tetrazolium compound by viable cells to generate a colored formazan product that is soluble in cell culture media. The final volume of 1.1 mL was pipetted to a 96-well plate with 100 μL per hole. The absorbance was determined in the 96-well for each time spot of 24 h, 72 h, and 144 h at OD = 490 nm. The MTS assay was repeated at least three times for each cell line and a time-point. For each experimental run, two or three identical samples were prepared and measured.The viability of cells was verified using an MTS calorimetric test. Briefly, fibroblasts were cultured in a multi-well plate (24 wells) in 1 mL of the corresponding culture medium. Next, 100 μL of MTS reagent (tetrazolium compound) was added to the cells in the culture medium. Then, cells were incubated at 37 °C in 95% air/5% COBefore fluorescence staining of actin filaments and the cell nucleus, cultured cells were pre-fixed to the substrate by adding a 1 mL solution of 3:7% of paraformaldehyde  to the culture medium for 2 min at 37 °C. Then, cells were washed with phosphate-buffered saline  3 times for 2 min. Afterwards, the sample was immersed in the solution of 3.7% of paraformaldehyde Fluka, Charlotte, NC, USA) for 15 min at room temperature, to fix cells firmly. After fixation, coverslips with cells were rinsed twice with the PBS buffer for 2 min. After that, a cold solution (4 °C) of 0.2% Triton X-100  was added for 4 min, followed by washing coverslips with the PBS buffer for 2 min. Next, cells were incubated with phalloidin conjugated with a 1:200 solution Alexa Fluor 488 dye  for 40 min, and subsequently, cell nuclei were incubated with a 1:5000 solution containing Hoechst dye (Sigma) for 14 min. The fluorescent images were collected from at least three repetitions carried out for each cell line and a time-point. For each experimental run, at least 10 fluorescent images from two or three coverslips with stained cells were collected.To elaborate on how fibroblasts behave in a distinct mechanical environment, two PDMS substrates characterized by 600 kPa (referred to here as a soft PDMS substrate) and 1.5 MPa (a stiff PDMS) were chosen. The spreading of fibroblasts was monitored by fluorescence imaging after 24h, 72 h and 144 h of culture .For stiff PDMS substrate (of 1.5 MPa), instead of spreading, cells tend to aggregate and to form agglomerates, with cells growing on each other, as shown in In our next step, we elaborate on how the elasticity of the PDMS substrates affects the proliferation of alive fibroblasts, corresponding to the viability of cells using an MTS assay applied at each time spot. The results are presented in The MTS values show that the proliferation of healthy lung fibroblasts a is signThe Young modulus of lung fibroblasts  was deteThe LL24 fibroblasts cultured on soft PDMS showed a significantly increased Young modulus after 72 and 144 h as compared to the stiff PDMS. These results differ noticeably from the observations made for the LL97A fibroblasts. Here, a significantly increased Young modulus on the stiff PDMS is observed at early incubation times (24 h), and for longer culture times , the stiffness of LL97A fibroblasts becomes comparable for both analyzed substrates.In addition to the mean elasticity, the spatial distribution of mechanical properties of cells was also analyzed, by recording the elasticity maps of cell fragments .A comparison of the elasticity maps recorded for a healthy LL24 cell line b and IPFThe possibility of discrimination between normal and fibrotic fibroblasts based on their chemical composition was verified using ToF-SIMS. Prior to the measurements, a drying protocol using multistep washing of cells in diluted solutions of anhydrous alcohol was applied .The representative m/z spectra, recorded for LL24 and LL97A cell  present 5H15NPO4+) identified as a major cholesterol [5H14NO+; m/z = 125.0, C2H6PO4+; m/z = 224.13, C8H19NPO4+), of phosphatidylcholine, which loaded PC1 positively.As seen in lesterol  and phoslesterol  fragmentFinally, the spatial distribution of cholesterol (m/z = 147) and phospholipids (m/z = 184) in cells were traced on composition maps, also provided by ToF-SIMS technique , showingOur study aimed to verify whether the physicochemical properties of lung fibroblasts, modified by substrate elasticity, can be used to discriminate between normal and fibrotic cells.The stiffness of the substrate is known to impact adhesion and proliferation of cells ,45,46,47On soft PDMS substrate (600 kPa), normal LL24, , as wellIn turn, on the stiff PDMS substrate (of 1.5 MPa), instead of spreading, cells tend to aggregate and to form agglomerates, with cells growing on each other, as shown in For healthy lung fibroblasts, the MTS tests show that the number of normal cells is reduced on the stiffer substrate. In contrast, for IPF-derived fibroblasts, the MTS analysis does not reveal any significant difference between the number of IPF-derived cells cultured on stiff and soft PDMS, concluded for fluorescence micrographs, indicating that substrate elasticity affects the arrangement of fibrotic cells rather than their ability to proliferate. The observed different response of both cell types to the substrate stiffness suggests that the internal structure of cells, modified for fibroblasts for ILD disorders, as well as the postulated alteration of their mechanical properties ,29, inflThe cytoskeleton organization, as well as the contractility of lung fibroblasts originating from IPF, differs from the normal lung tissue . KnowingFor both cell lines, an increase of Young’s modulus with time is observed, especially for cells cultured on softer PDMS substrate. Here, elasticity modulus doubles for normal fibroblasts and becomes four times larger after 144 h of incubation for IPF-derived ones. This effect may be related to the presence of neighboring cells, which affects Young’s modulus even for AFM measurements carried out on individual cells. The recorded moduli imply that normal fibroblasts remain more rigid as compared to their fibrotic counterparts. Observed difference may indicate the impairments of IPF-derived fibroblasts induced by the fibrosis process, but it can be also linked with the donor age, different for both cell lines. To resolve this issue, extended studies for patient-derived fibroblasts are required.Observed values of moduli are much higher than the physiological range of the Young modulus of pulmonary tissue (~2–10 kPa) reported elsewhere ,25,57,58The mechanical properties of cells are substantially different for the cellular membrane and the subcellular components, such as the cytoskeleton or nucleus . The memThe observed slight differences in elastic properties between normal and IPF-derived fibroblasts, corresponding to the internal arrangement of the cytoskeleton, may influence the mechanical properties of the examined cells, which may be therefore considered as potential diagnostic markers.Inflammatory lung diseases are known to affect the biochemical properties of the tissue. Several reports suggest the critical importance of lipoproteins and cholesterol for normal lung physiology , howeverTaking into account the biochemical changes occurring at the cell surface, we test the possibility of discrimination between normal and fibrotic fibroblasts, based on their chemical composition using ToF-SIMS. The representative m/z spectra, recorded for LL24 and LL97A cell , presentIn turn, ToF-SIMS analysis does not show any difference in the intensities of Ca (m/z = 39.96) and CaO (m/z = 56.96) peaks recorded for both cell lines, indicating a similar amount of Ca for them , which iFinally, the spatial distribution of cholesterol (m/z = 147) and phospholipids (m/z = 184) in cells was traced on composition maps, also provided by ToF-SIMS technique .In our previous work, we have shown, that the stiffness of the substrate, changing from 1.5 MPa to 600 kPa, significantly affects the behavior of both normal and IPF-derived lung fibroblasts, indicating that elastic moduli of cells may play an important role in the development of lung fibroblasts also in the elasticity range far from the physiological one. This motivated us to expand the study and test in details the impact of substrate stiffness on physicochemical properties of normal (LL24) and IPF-derived (LL97A) lung fibroblasts. The growth and viability of cells were recorded using fluorescence microscopy and MTS tests respectively, for culture times ranging from 24 h up to 144 h. For normal fibroblasts, both fluorescence imaging and MTS tests show that the number of normal cells is significantly reduced on the stiffer substrate. In contrast, for fibrotic fibroblasts, MTS analysis does not reveal any remarkable difference between the number of IPF-derived cells cultured on stiff and soft PDMS, whereas fluorescence images show a significant variation of their density. These results indicate that substrate elasticity affects the arrangement of fibrotic cells rather than their ability to proliferate. Additionally, the measurements of elastic modulus of cells, performed using atomic force microscopy working in a force spectroscopy mode showed that normal fibroblasts remain more rigid as compared to their fibrotic counterparts. Observed difference may indicate the impairments of IPF-derived fibroblasts induced by the fibrosis process, but it can be also linked with the donor age, different for both cell lines. To resolve this issue, extended studies for patient-derived fibroblasts are required. Moreover, elasticity maps show that the spatial distribution of mechanical properties differs for both examined cell lines. The chemical properties of normal and IPF-derived lung fibroblasts were inspected using time-of-flight secondary ion mass spectrometry (ToF-SIMS), combined with principal component analysis (PCA), which allows to detect even subtle differences between the samples. As a result, significant differences in the distribution of cholesterol and phospholipids were recorded for healthy and IPF-derived fibroblasts, whereas no differences in Ca abundance were observed.The observed different response of both cell types to the substrate elasticity suggest that the internal structure of cells, modified for fibroblasts for ILD disorders, as well as the postulated alteration of their mechanical properties influence the cellular behavior and adaptability to the external conditions and might be therefore be seriously considered as an important factor while constructing a diagnostic tool, enabling the fast and reliable identification of IPF fibroblasts. However, before applying such a diagnostic tool for a wide use in medical laboratories, a special protocol for the collection of cells from patients’ tissue must be developed. Although the proposed diagnostic method is not straightforward, we believe that it is worth considering, as nowadays, there is no fast and reliable diagnostic method enabling easy recognition of IPF, which is very often misidentified with other Interstitial lung diseases (ILDs), especially NSIP. Therefore, in our opinion, it seems to be very perspective, as it enables univocal, easy, cheap, and relatively fast diagnosis. Even with the separate step of cell line derivation from the patient’s tissue, the diagnostic process should not exceed a few weeks, which is significantly shorter than the current procedures. Moreover, it is based on the analysis of cell stiffness, which is a measurable, objective parameter, and therefore reduces the possibility of the incorrect interpretation of the results.


The aim of this is to identify the possible regulatory mechanisms mediated by the polyamines on sperm-specific channels under capacitation and non-capacitation conditions. The results showed that the presence of polyamines did not directly influence the activity of calcium and chloride channels. However, the results suggested an interaction of polyamines with sodium and potassium channels, which may contribute to the membrane Vm during capacitation. In addition, alkalization of the pHi revealed the possible activation of sperm-specific Na+/H+ exchangers (NHEs) by the increased levels of cyclic AMP (cAMP), which were produced by soluble adenylate cyclase (sAC) and interact with the polyamines, evidence that is supported by in silico analysis.Polyamines are ubiquitous polycationic compounds that are highly charged at physiological pH. While passing through the epididymis, sperm lose their capacity to synthesize the polyamines and, upon ejaculation, again come into contact with the polyamines contained in the seminal fluid, unleashing physiological events that improve sperm motility and capacitation. In the present work, we hypothesize about the influence of polyamines, namely, spermine, spermidine, and putrescine, on the activity of sperm channels, evaluating the intracellular concentrations of chloride [Cl Furthermore, we have evaluated the effect of the polyamines on Vm and pHi under capacitation and non-capacitation conditions. Following our hypothesis that polyamines may influence the activity of sAC, we performed an in silico analysis with polyamines and sAC. In the present work, we hypothesize that polyamines may influence the activity of sAC, which in turn modulates ion homeostasis by the alkalization of pHi. To test this hypothesis, we evaluate the effects of spermine, spermidine, and putrescine on the intracellular concentrations of chloride i was evaluated. The results showed that the presence of putrescine induces a significant increase of the Cl− influx under capacitation conditions .After selecting the optimal polyamine concentration, i increases [−]i induced by the putrescine (−]i (3 present in the capacitation media or via the possible interaction of polyamines with casein kinase II (CK2), which also modulates the activity of CFTR. CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. While the MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls the channel-gating properties i only in the presence of putrescine  family, which are highly effective at regulating pHi. These cation exchangers constitute a family of membrane proteins commonly named Na+/H+ exchangers (NHEs) i ,36, a lof [Na+]i ,37. Thesf [Na+]i , and polcitation . The presporters ,40. H+ cs (NHEs) . In mouss (NHEs) ,39. Thers (sACs) . On the s (sACs) ,44,45. Ts (sACs) ,46,47. Ms (sACs) . 2+]i and [Cl−]i appear to be independent of the presence of polyamines during capacitation. However, the results of this work suggest the interaction of polyamines with sodium and potassium channels, which may contribute to the membrane Vm during capacitation. In addition, the alkalization of pHi suggests a possible activation of NHEs by the increased levels of cAMP produced by sAC, which potentially interacts with the polyamines. Finally, as a perspective of this work, we will attempt to relate the activity of sAC with the variation of polymines levels in seminal fluid, which is possibly found in infertile men and patients with diabetes.In conclusion , the inc4(3), which was purchased from Biotium Inc. . Fura-2 AM, CoroNaTM Green, MQAE, BCECF-AM, and the FluxOR potassium ion channel assay were purchased from Thermo Fisher, Scientific .All reagents were purchased from Sigma Chemical Co.  or Mallinckrodt Baker , except for DiBACAdult male mice  were obtained from the ENCB-IPN Vivarium, weighing from 20 to 25 g. They were maintained in polypropylene cages at room temperature, with a normal light/dark cycle, and provided standard commercial food and water ad libitum. The investigation and animal protocols were approved by The Institutional Ethics Committee of Escuela Nacional de Ciencias Biológicas of the Instituto Politécnico Nacional (protocol no. 902144 in Januaty the 7th of 2014) in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health .3, 0.36 mM NaH2PO4, 0.49 mM MgCl2, 0.25 mM sodium pyruvate, 20 mM sodium lactate) 1.8 mM CaCl2, 5.56 μM glucose, and 1 mg/mL albumin to achieve capacitation conditions (capacitation medium). The same medium was modified by replacing 11.9 mM NaHCO3 with 25 mM HEPES and removing glucose and albumin to achieve non-capacitation conditions (non-capacitation medium). The final sperm concentration for each experiment was 8–10 × 106 spermatozoa/mL. Experiments were performed in triplicate at 37 °C  located at the bicarbonate binding site . The 5D0p < 0.05.Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). All data are shown as the mean±SEM. Statistical significance of the results was determined using one-way ANOVA followed by Dunnett´s test. Differences were considered significant at


Orofacial clefts (OFCs) comprise a wide range of malformations, including cleft lip, cleft palate, and cleft lip with cleft palate, which can vary in terms of etiology, severity, and disease burden.This study aimed to evaluate the correlation between various risk factors and orofacial cleft disorder spectrum in newborns.A total of 323 cases and 400 controls were enrolled in this study and evaluated in terms of the maternal history of abortion or miscarriage, child’s sex, maternal and paternal age, maternal history of systemic disease, history of medication therapy during pregnancy, birth order, consanguineous marriage, and complications during pregnancy.P < 0.05). However, the analyses revealed that the other variables could not potentially increase the risk of OFCs (P > 0.05).Analysis of the results suggested that consanguineous marriage, a maternal history of abortion/miscarriage, and complications during pregnancy could potentially increase the risk of OFCs in children (Multiple cofactors may simultaneously contribute to the formation of such abnormalities; therefore, a comprehensive, multidisciplinary care program is necessary to ensure a successful pregnancy period and the birth of a healthy newborn. Orofacial clefts (OFCs) comprise a wide range of malformations, including cleft lip, cleft palate, and cleft lip with cleft palate, which can vary in terms of etiology, severity, and disease burden . The etiThis retrospective, case-control study was conducted at the Craniofacial and Cleft Research Center of Isfahan University of Medical Sciences, Isfahan, Iran, from 2014 to 2020. Data were collected by reviewing the medical records of mothers, fathers, and children, with or without a reported history of OFCs. The study procedures were verbally explained to the parents, and written consent was obtained. All procedures in this study were in accordance with the ethical standards of the local research committee, as well as the Declaration of Helsinki.Mothers who received childbirth care at the hospital and delivered newborns with or without OFCs were included in this study. The exclusion criteria were as follows: (1) Patients without medical records, (2) patients with incomplete medical records, (3) mothers not receiving medical care at the hospital during 2014–2020 and also, (4) patients diagnosed with syndromic OFCs were excluded. The study variables included the maternal history of abortion or miscarriage, child’s sex, maternal and paternal age, maternal history of systemic disorders, history of medication therapy during pregnancy, birth order, consanguineous marriage, and complications during pregnancy.P values less than 0.05 were considered statistically significant.The obtained data were stored in a database and analyzed using SPSS® Version 26.0 . Statistical analyses were carried out using logistic regression analysis. To estimate the risk of OFCs, the odds ratio (OR) was calculated at a confidence interval of 95% (95% CI). A total of 323 cases and 400 controls were enrolled in this study. The mean ± SD of age was 31.4 ± 5.4 and 30.78 ± 5.05 years in the case and control mothers, respectively. Also, the mean ± SD of age was 32.60 ± 4.80 and 31.69 ± 4.76 years in the case and control fathers, respectively. Overall, 171 (53%) children in the case group and 204 (51%) children in the control group were female. Based on the results, 42 (13%) mothers of children with OFCs and 28 (7%) control mothers reported a history of abortion or miscarriage. Also, 119 (36%) mothers of children with OFCs and 93 (23%) control mothers reported a consanguineous marriage.A review of mothers’ medical records showed that 36 (11%) mothers of children with OFCs and 32 (8%) control mothers suffered from at least one systemic disorder. Medication therapy during pregnancy was reported by 122 (38) case mothers and 124 (31%) control mothers. The results showed that 92 (29%) mothers of children with OFCs and 88 (22%) control mothers experienced a complication during pregnancy.P > 0.05). However, the birth of a child with OFCs had a potential correlation with the maternal history of abortion/miscarriage , consanguineous marriage , and complications during pregnancy  (P > 0.05).Based on the results, the correlation between the birth of a child with OFCs and child’s sex , medication use during pregnancy , and history of a systemic disorder  was not significant , which is consistent with the results of several studies [Our findings revealed a potential correlation between the history of abortion/miscarriage and the birth of a child with OFCs in the subsequent pregnancies . The male/female ratio in our study was about 0.88, whereas this ratio in another study conducted in Iran was 2.3 [The results of the present study showed that female newborns were slightly more likely to develop cleft lip and cleft palate, compared to males, although this correlation was not significant . However, the independent analysis of medications used by mothers during pregnancy was beyond our capacity. Our investigations showed that the most common medications used by the mothers of children with OFCs during pregnancy included anti-stress medications, miscarriage prevention drugs, antibiotics, and some pain-relief medications, such as non-steroidal anti-inflammatory drugs. Nevertheless, the results of various studies [Moreover, medication use during pregnancy has been identified as a factor, which cannot potentially increase the risk of OFCs in children . Similarly, a study by Bui et al. [P > 0.05) [Pregnancy complications such as maternal cold or flu, preeclampsia, anemia, pregnancy hypertension, gestational diabetes, and intrauterine hypoxia can potentially increase the risk of OFCs in children  .P > 0.05). Similarly, the results of a study by Mirilas et al. [According to the definition of systemic disorders, which are known to affect a number of organs and tissues or the body as a whole , we founs et al.  showed ts et al.  and Kraps et al. , in sepaP < 0.05). However, the results of studies by Mbuyi-Musanzayi et al. [α = 0.0185), which is significantly higher than the global average [In the present study, the most remarkable factor, which potentially increased the risk of OFCs, was consanguineous marriage , compared to Western developed countries, where this type of marriage is not very common. The implicit conclusion that can be drawn from these findings is that studies conducted in different countries can only identify the factors, which may increase the risk of OFCs. However, to reach a reliable understanding of the factors increasing the risk of OFCs in a specific society, it is essential to conduct an exclusive study of risk factors in the target population.In the present study, considering the wide range of OFC abnormalities with multiple underlying factors, it was not possible to examine all factors influencing OFCs due to the lack of facilities; this can be considered as the most important limitation of our study. Also, examination of different trimesters, a separate examination of different types of OFCs, paternal factors, smoking, consumption of alcohol, socioeconomic status of families, the status of maternal education, and maternal body mass index was among the crucial factors that could not be addressed in this study and require further investigation.Our results indicated a potential correlation between some variables such as consanguineous marriage or a maternal history of abortion/miscarriage and the birth of a child with OFCs in the subsequent pregnancies. Since the formation of OFC, lesions are not usually dependent on a single factor, and multiple cofactors may simultaneously contribute to such abnormalities, and a comprehensive, multidisciplinary care program is necessary to ensure a successful pregnancy period and birth of a healthy newborn. It is also necessary to conduct further studies in various populations in order to gain a comprehensive understanding of the multifactorial etiology of the OFCs disorder spectrum.


At least one ultrasound is recommended to predict fetal growth restriction and low birthweight earlier in pregnancy. However, in low-income countries, imaging equipment and trained manpower are scarce. Hence, we developed and validated a model and risk score to predict low birthweight using maternal characteristics during pregnancy, for use in resource limited settings. We developed the model using a prospective cohort of 379 pregnant women in South Ethiopia. A stepwise multivariable analysis was done to develop the prediction model. To improve the clinical utility, we developed a simplified risk score to classify pregnant women at high- or low-risk of low birthweight. The accuracy of the model was evaluated using the area under the receiver operating characteristic curve (AUC) and calibration plot. All accuracy measures were internally validated using the bootstrapping technique. We evaluated the clinical impact of the model using a decision curve analysis across various threshold probabilities. Age at pregnancy, underweight, anemia, height, gravidity, and presence of comorbidity remained in the final multivariable prediction model. The AUC of the model was 0.83 . The decision curve analysis indicated the model provides a higher net benefit across ranges of threshold probabilities. In general, this study showed the possibility of predicting low birthweight using maternal characteristics during pregnancy. The model could help to identify pregnant women at higher risk of having a low birthweight baby. This feasible prediction model would offer an opportunity to reduce obstetric-related complications, thus improving the overall maternal and child healthcare in low- and middle-income countries. Low birthweight (LBW), a weight at birth of less than 2500 g (5.5 lb), continues to be a significant public health problem globally. It is estimated that 15% to 20% of all births worldwide are LBW, accounting for more than 20 million in a year ,4,5,6, sLBW or being small for gestational age increases infant morbidity and mortality ,10,11,12Demographic factors such as young maternal age, higher birth order, prim-gravida, low educational level, and poor maternal nutritional status before and during pregnancy are well recognized risk factors for LBW ,23,24,25LBW has a remarkable impact on the political, social, economic, and healthcare system in LMICs. Hence, by the end of 2025, the World Health Assembly set a policy target to reduce LBW by 30% . StrategThe present study used data from the Butajira Nutrition, Mental health and Pregnancy (BUNMAP) project in Ethiopia, a population-based cohort established in 2016. It is a cohort of pregnant women and their offspring living in selected clusters of Butajira Health and Demographic Surveillance Site (HDSS), South Ethiopia. Butajira HDSS is one of the oldest surveillance sites in Africa established in 1986. The livelihood of the residents is based on subsistent farming. Khat (Catha edulis Forsk) and chili peppers are the main cash crops, while maize, banana, and Ensete (Enseteventricosun) are the main staples. The cohort is still ongoing and by the time of this analysis, 881 pregnant women were enrolled and planned to follow the mother-child pair up to the third birthday of the child. Among those enrolled, 388 gave birth, whereas the remaining (493) were in first (245), second (156), or third (92) trimester of pregnancy at the time of this analysis (May 2019). Ethical clearance was obtained from the Institutional Review Boards (IRB) of Addis Ababa University, College of Health Sciences (code: 099/17/SPH). Written informed consent and parental assent were obtained from study participants after explaining the possible risks, benefits, purposes of the study, issues of confidentiality and voluntarism. The study is in compliance with the principles of the declaration of Helsinki. All data were stored either in password-protected computers or, in the case of paper records, in locked files in the project’s locked office in Addis Ababa, Ethiopia. The theoretical design of the present study was; the incidence of low birthweight (at time 1) as a function of multiple predictors during pregnancy (time 0). The source population for the cohort were all 15 to 49 years old women living in Butajira HDSS, who have the capacity to be pregnant. All pregnant women who were enrolled into the cohort and fulfilled the eligibility criteria were included in the analysis. To be included in this study, women must meet all of the following eligibility criteria; (1) should gave birth and (2) birthweight was taken within 72 h of delivery. Whereas, women with fetuses having congenital malformations during the ultrasonographic evaluation or twins or above pregnancies were excluded. Out of 388 pregnant women who gave birth, for 8 of them birthweight was not taken within 72 h of delivery and 1 delivered for twin babies, making our final sample 379. Outcome Assessment. After enrollment, three ultrasound examinations of pregnant women were done, one during each trimester, to estimate gestational age, intrauterine fetal growth, and presence of any congenital anomaly. Birthweight was taken within 72 h of post-delivery using a digital scale. The main outcome, LBW, was defined as a weight of neonate below 2500 g (5.51 pounds).Predictor assessment. A questionnaire was adapted from the Ethiopian Demographic and Health Survey and other relevant literatures. A range of socio-demographic, obstetric, and clinical characteristics of the women including, morbidity, educational status, marital status, occupation, gravidity, parity, ANC utilization, family planning, and the interval between pregnancies were collected. Nutritional status including height, and weight was taken for all women at baseline and during each trimester of pregnancy. The level of anemia was also assessed by measuring hemoglobin in red blood cells, using a Hemo-Cue (Hb-201) instrument. Training was given for data collectors and supervisors about the objective of the research, how they will collect the data, keep the collected data, and supervise the data collection process. Afterward, a pilot study was done in order to assure that data collectors and supervisors are competent enough to collect and supervise the data collection process. In the case of paper form, questionnaires were controlled for completeness and logical errors, and where errors were found, the questionnaires were redone. The data were collected using the Open Data Kit (ODK) platforms and were exported to the R statistical programming language version 3.6.0 for further processing and analysis. There was 8 (2.1%), 7 (1.8%), and 6 (1.6%) missing values for hemoglobin level, weight, and height measurements. Moreover, marital status, alcohol consumption and the presence of chronic morbidity each had 1 (0.3%) missing values . We assup-value < 0.25 in the univariable analysis to the multivariable model to be more liberal. Afterward, we used a stepwise backward elimination technique with p-value < 0.10 for the likelihood ratio test to fit the reduced model. As the pregnant women came from different clusters, individual data were likely to be clustered within the different kebeles, which could affect the association of the predictors with the low birthweight. We accounted for such possible non-random differences within kebeles (clusters) using multilevel logistic regression techniques [We performed a univariable analysis using logistic regression to obtain insight into the association of each potential determinant with LBW and to select potential predictors for multivariable analysis. We fit all the variables with chniques ,34. We uTo check for the model accuracy, we computed the area under the ROC curve (discrimination) and calibration plot  using “classifierplots” and “givitiR” packages of R respectively ,36. The To evaluate the clinical and public health impact of the model, we performed a decision curve analysis (DCA) , of stanTo construct an easily applicable low birthweight prediction score, we transformed each coefficient from the model to a rounded number by dividing to the lowest coefficient. The number of points was subsequently rounded to the nearest integer or half-integer. We determined the total score for each individual by assigning the points for each variable present and adding them up. The predicted probability of LBW was presented according to three categories of the risk score for reasons of statistical stability and practical applicability. The categories were arbitrarily chosen with a view to reasonable size of each category as well as public health sensibility. Later, the score was transformed to a dichotomous “prediction test,” allowing each pregnant woman to be classified as at high or low risk of LBW. We carried out a sensitivity analysis around different cutoff points of 3, 3.5, 4, 4.5, 5, and 5.5. The sensitivity, the specificity, the positive and negative predictive values, and the likelihood ratios of categorized values of the score were calculated .This study was reported in accordance with the TRIPOD  statement , which iWe included a total of 379 women who gave birth and birthweight was taken within 72 h of delivery. P < 0.25 in the univariable analysis were; age at current pregnancy, BMI, height, educational status, hemoglobin level, attending previous ANC, gravidity, and presence of comorbidity. Then, six predictors remained in the reduced multivariable regression analysis; younger age (<20 years), underweight (BMI < 18.5), short stature (height < 155cm), anemia (hemoglobin < 11mg/dl), primi-gravida, and presence of comorbidity. Using the results, a prediction model was developed and equation for the prediction model was obtained.  were low birthweight infants. The mean birthweight was 2788.4 g (SD: 611.4). After review of literature, 13 demographic, obstetric, and clinical characteristics of the mother were collected at baseline and considered to predict low birthweight at term. The univariable analysis found several factors were eligible to be included in the prediction model. Variables with btained. p-value of 0.89, indicating that the model does not misrepresent the data  a. The cathe data b. ValidaUsing the coefficients (β) the predicted risk cutoff point was a probability of > 0.2631, with sensitivity of 71% (95%CI: 60–81), specificity 82% (95%CI: 77–86), positive predictive value 52% (95%CI: 43–62), and negative predictive value of 91% (95%CI: 87–94). The positive and negative likelihood ratios were 3.9 (95%CI: 2.95–5.14) and 0.35 (95%CI: 0.25–0.50), respectively. As shown in For practical utility, we developed a simplified risk score from the model. Rounding of all regression coefficients in the reduced model resulted in a simplified prediction score presented in When dichotomized to high risk (>4) and low risk (≥4) based on the risk score, 114 (30.1%) were categorized as high risk and 265 (69.9%) as low risk for LBW. Using “Youden index”, the suggested cutoff to predict LBW using risk scores is > 4 with a sensitivity of 72.3% (95%CI: 61–82), specificity of 81.8% (95%CI: 77–86), positive predictive value of 52.6% (95%CI: 43–62), negative predictive value of 91.3% (95%CI: 87–94), positive likelihood ratio of 3.96 (95%CI: 3.01–5.22), and negative likelihood ratio of 0.34 (95%CI: 0.24–0.48). Detailed information on the risk score performance at different possible cutoff points is available in Annex 2.The present study shows the incidence of low birthweight was 21.9%. The optimal combination of maternal characteristics to predict LBW are age < 20, BMI < 18.5, hemoglobin < 11 mg/dl, height < 155cm, prim-gravida, and presence of comorbidity. This study quantified the predictive performance of a model using maternal characteristics during pregnancy without any advanced laboratory or imaging tests. Predicting the probability of LBW in pregnant women is essential to take appropriate measures accordingly. The WHO recommends one ultrasound for every pregnant women before 24 weeks of gestation to estimate gestational age, fetal weight, and any fetal anomalies . NeverthIn our prediction score, using 4 as cutoff point has an acceptable level of specificity, sensitivity, PPV, and NPV to predict LBW. It is also possible to shift the cutoff point to increase either of the accuracy measures depending on the program aim and availability of resources. Although the ultrasonographic evaluation of pregnant women gives a better indicator of fetal growth and prediction of birthweight, maternal characteristics during pregnancy alone enabled to predict the risk of low birthweight in advance. Our prediction model is not a replacement for the ultrasonographic assessment of pregnant women; however, it will be a screening tool in resource-poor settings for further diagnostic workup and management options. The simplified risk score derived from the regression models is easier to use in routine clinical and public health practice than the regression models and has comparable discrimination and calibration.This study has several strengths. Firstly, we used an adequate number of participants with the outcome, i.e., LBW, which helped us to construct the model using a sufficient number of predictor variables. Secondly, we internally validated our model using bootstrapping technique and resulted small optimism coefficient, indicating our model is less sample dependent. Thirdly, our prediction model is constructed from easily obtainable maternal characteristics that make it applicable in primary care settings. However, the findings from this study should be interpreted with the perspective of the following limitations. As a single site study, it is confined to a single area, which needs external validation before using it in another context. Due to small sample size, we did not validate the model in separate datasets. However, the bootstrapping showed minimal optimism, indicating a stable predictive capability of the model. Lastly, since the data were from research setting, where training was given to data collectors, some deviation in data quality was expected in real-world practice. Nevertheless, the predictors included in the model are easy to measure, which indicates the impact on the accuracy of the model in clinical or public health practice is minimal. The model will provide its maximum benefit provided that all the required predictor information is collected. This study shows the possibility of predicting LBW using a simple prediction model constructed from maternal characteristics. The prediction score will help to do a risk stratification of pregnant women and to identify those at higher risk of having an LBW baby. Subsequently, high-risk groups can be linked to a center, which is equipped with ultrasound facilities for further assessment and better management during pregnancy, delivery, and post-natal period. Hence, this feasible prediction score would offer an opportunity to reduce neonatal complications related with low birthweight and thus improving the overall maternal and child healthcare. We strongly recommend validating the prediction tool in another context before introducing it to the clinical and public health practices, preferably using real-world data.


Stroke is emerging as a significant health issue that threatens human health worldwide and as a common sequela of stroke spastic paralysis after stroke (SPAS) has received wide attention. Currently, several systematic reviews have suggested that the commonly used acupuncture therapy  has achieved significant efficacy in the treatment of SPAS. In this study, network meta-analysis will be used to analyze the results of different clinical trials and evaluate the differences in the efficacy of different acupuncture treatments for SPAS.Only randomized controlled trials will be included and all patients were diagnosed as spastic paralysis after stroke. A computer-based retrieval will be conducted at CNKI, WanFang databases, VIP, Sinoed, Pubmed, Embase, Web of Science, and the Cochrane library. The search period limit is from the time the date of database establishment to April 17, 2020. To avoid omissions, we will manually retrieve relevant references and conference papers. The risk of bias in the final included studies will be evaluated based on the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions. All data analysis will be conducted by Revman5.3, WinBUGS 1.4.3, and Stata14.2.This study quantified the effectiveness of each intervention for different outcome indicators. The primary outcomes include the Fugl-Meyer Assessment score, the modified Ashworth scale for the assessment of spasticity, and Barthel Index. The secondary outcomes include clinical effectiveness and adverse reactions.It will provide evidence-based medical evidence for clinicians to choose more effective acupuncture therapy for SPAS Relevant articles revealed the possible etiologies that mainly injured the upper motor neurons, resulting in a significant increase in muscle tension.,7 If failure to take active treatment may cause Joint deformation, muscular atrophy, and pain, which may affect the rehabilitation of the limbs and the quality of life.Stroke is emerging as a significant health issue that threatens human health worldwide. Besides a previous study shows stroke is a leading cause of death in China.,10 However, concerns have been raised about the side effects of the drugs, the invasive nature of the surgery, and the high cost of treatment. Due to limitations of current SPAS treatment, it is particularly important to explore alternative treatment options with better efficacy, fewer side effects, and low price. As a part of traditional Chinese medicine treatment, acupuncture has been used to treat stroke patients for many years in China and become more popular in Western countries in recent years. Modern research shows that acupuncture can enhance immunity, regulate blood circulation, relieve the pain, help muscles relax and exercise more passively, thereby increasing recovery.–14Nowadays, the common treatment methods for SPAS are medications, surgical interventions, and physical therapy.–19 But traditional systematic reviews can only directly compare 2 interventions, and cannot compare multiple acupuncture therapies. At present, there are various types of acupuncture, the 4 acupuncture therapies , warm acupuncture (WA), filiform needle acupuncture) analyzed in this study are commonly used methods. Meanwhile the advantages of various acupuncture therapies are not the same, so the choice of which acupuncture therapy can bring greater curative effect has brought troubles to clinical operators. In this study, the effectiveness of acupuncture therapy commonly used in clinical will be ranked by network meta-analysis, to provide scientific evidence-based medicine basis for the clinical selection of the best.More and more systematic reviews suggest that acupuncture plays an important role in patients with SPAS.2 The protocol of the systematic review has been registered in the INPLASY website(registration number is INPLASY202050058). If there are any adjustments during the entire study period, we will fix and update the detailed information in the final report in time.This systematic review protocol will be reported strictly adherence to the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P).33.13.1.1Only randomized controlled trials (RCTs) will be included and no restrictions on language. Non-RCTs such as system reviews, meeting abstracts will be excluded. Interventions incompatible with the inclusion criteria and animal experiments will be excluded. Duplicate publications are preferred to the 1 with the most recent and comprehensive data.3.1.2All adult patients are diagnosed as SPAS. There will also be no restrictions based on gender, race, and the course of the disease. However, the following patients will be excluded:(1)Patients with mental illness who cannot cooperate with treatment.(2)Patients with complications or vital organ failure.(3)Pregnant or lactating women.(4)Patients with adverse reactions or pain that cannot withstand acupuncture.3.1.3The experimental group only use EA, FA, WA or milli-acupuncture, and the control group uses rehabilitation or a comparison of the above 4 acupuncture methods. Patients in both groups could receive conventional medical treatment and treatment duration and frequency are not limited.3.1.4–23 The secondary outcomes include clinical effectiveness and adverse reactions. Clinical effectiveness including 4 grades of recovery, obvious effect, effective and ineffective. Effective rate =  × 100%.The primary outcomes include the Fugl-Meyer Assessment score (FMA), the modified Ashworth scale (MAS) for the assessment of spasticity, and Barthel Index (BI). These indexes can evaluate the recovery of limb spasm.3.2A computer-based retrieval will be conducted at CNKI, WanFang databases, VIP, SinoMed, Pubmed, Embase, Web of Science, and the Cochrane library. The search period limit is from the time the date of database establishment to April 17, 2020. To ensure the comprehensiveness of the search, relevant references and conference literature are also included. A search strategy is as follows:(((((((([stroke [MeSH Terms]) OR ) OR ) OR (sequelae)) OR (spasm)) OR ) OR (spastic hemiplegia)) AND ((((((((acupuncture) OR (EA)) OR (FA)) OR (WA)) OR (filiform needle acupuncture)) OR (warm needle)) OR (fire-needle)) OR (needle acupuncture))) AND  OR “RCT” [Publication Type]) OR “clinical trials” [Publication Type])).3.3Two evaluators (RQW and RFX) will independently screen all relevant literature according to the inclusion and exclusion criteria. Then import them into Endnote for centralized management. We then go through the title abstract and the full text, in turn, to determine which study ultimately meets the criteria. During this process, if there is any dispute, the decision will be made by the third evaluator (JWH). Also, we will establish a document information extraction table in EXCEL, the extracted data includes the following: title, author, publication time, abstract, sample size, number of cases in each group, age, gender, course of the disease, distribution method, intervention, treatment course, outcome indicators, and so on.3.4,25 This work will also be done independently by 2 reviewers.The risk of bias in the final included studies will be evaluated based on the guidelines of the Cochrane Handbook for Systematic Reviews of Interventions. The evaluation criteria include 7 items: selections bias, performance bias, detect bias, attrition bias, reporting bias, and other bias. Each item will be graded into 3 levels: “high risk”, “low risk”, and “not clear”.3.5,27 In the WinBUGS software, Bayesian network meta-analysis is performed by the Markov Chain Monte Carlo (MCMC) method, which is simulated by 4 chains, the number of iterations is set to 50,000, and the step size is set to 10. At the same time, the potential scale reduction factor (PSRF) is used to evaluate the convergence of the results. When 1.00≤PRSF≤1.05, it indicates that the results converge well and the results obtained are highly reliable.Revman 5.3 software is used for bias evaluation and traditional Meta-analysis. For continuous variables , the results will be reported as mean difference (MD) with 95% confidence interval (CI); Count data  will be calculated with the odds ratio (OR) and 95% CI. WinBUGS 1.4.3 and Stata 14.2 are used for network meta-analysis. The SUCRA value ranges from 0 to 100. The larger the value, the intervention is considered to have better efficacy.We can calculate the surface under the cumulative ranking curve (SUCRA) to estimate the possible ranking order of various interventions.3.6P-value. At the same time, the node split model will be used to judge whether each node has local inconsistency. If P > .05, there is no significant inconsistency, and the consistency model is used. As for the results obtained from the consistency model analysis, the stability of the results can be tested by the inconsistent model .,33Because of the large number of interventions involved in this study, the Loop inconsistency test of studies with direct and indirect evidence is needed in the evidence network for each outcome indicator. Calculate the inconsistency factor (IF), and judge whether there is inconsistency according to the size of IF value and 3.7I2 and P values. For the test results with obvious heterogeneity, the source of heterogeneity should be analyzed. Subgroup analysis can be conducted according to the different sources of heterogeneity, such as the following aspects: treatment duration, disease course, underlying disease, race, gender, age, and so on. If no clear source of heterogeneity can be found, only descriptive analysis can be conducted.The heterogeneity between trials is quantified with the  Then, the strength, reliability, and stability of the results will be analyzed by observing the heterogeneity of different tests and whether the combined results changed after various treatments.The purpose of sensitivity analysis is to eliminate low-quality studies and different statistical models.3.8 If there is a difference in symmetry or distribution, there will be a publication bias or a small sample effect.If the outcome indicators include in study ≥10, funnel plots will be used to assess the publication bias of the included trials.3.9Due to this is a protocol for systematic review and network meta-analysis, all data of this study are from published studies and do not involve patients, so ethical approval will not be necessary. The findings of this study will be disseminated to a peer-reviewed journal and presented at a relevant conference.4With the continuous development and improvement of acupuncture technology, there are a variety of acupuncture treatments that are considered to be effective in the treatment of SPAS. However, the therapeutic advantages of different acupuncture therapies are different from each other, which confuses clinical choices. Network meta-analysis overcomes the shortcomings of traditional systematic reviews and can integrate direct and indirect evidence. Besides, it provides an intuitive comparison of the efficacy and safety of available technologies. So the results can provide clinicians and patients with the ability to determine the best treatment options. We will use the network meta-analysis method to assess the effectiveness of 4 commonly used clinical acupuncture manipulations, and quantified their effectiveness according to different outcome indicators.Conceptualization: Ruiqi Wang, Chunhua Huang, Rongfang XieData curation: Ruiqi Wang, Chunhua Huang, Jinwen HuFormal analysis: Ruiqi Wang, Chunhua Huang, Rongfang XieProject administration: Ruiqi Wang, Chunhua Huang, Qingzhong WuSupervision: Ruiqi Wang, Chunhua Huang, Wangfu RaoWriting – review & editing: Ruiqi Wang, Chunhua Huang, Rongfang Xie


Papaver rhoeas (Papaveraceae), as a result of its introduction into Central Europe and the impact of those modifications on its interactions with pollinators. We found that while flowers of Eastern Mediterranean poppies reflect exclusively in the red part of the spectrum, those of Central European poppies reflect both red and ultraviolet (UV) light. This change coincides with a shift from pollination by glaphyrid beetles (Glaphyridae) to bees. Glaphyrids have red-sensitive photoreceptors that are absent in bees, which therefore will not be attracted by colors of exclusively red-reflecting flowers. However, UV-reflecting flowers are easily detectable by bees, as revealed by visual modeling. In the North Mediterranean, flowers with low and high UV reflectance occur sympatrically. We hypothesize that Central European populations of P. rhoeas were initially polymorphic with respect to their flower color and that UV reflection drove a shift in the pollination system of P. rhoeas that facilitated its spread across Europe.Evolutionary change is considered a major factor influencing the invasion of new habitats by plants. Yet, evidence on how such modifications promote range expansion remains rather limited. Here we investigated flower color modifications in the red poppy,  A major environmental perturbation induced by dispersal of plants to new habitats is the decoupling from their native pollinators. The lack of pollinators can induce pollen limitation, constraining the spread of plants into new environments ,2. WithiPapaver rhoeas, is an iconic plant whose cultural relevance can be traced back several millennia. Its ornamental, pharmacological, and edible value was already recognized as early as the 18th dynasty in Egypt (1504–1450 BC) [P. rhoeas originated in the East Mediterranean [P. rhoeas successfully established self-perpetuating populations  in human-made habitats across Europe [The red poppy, 1450 BC) . Taxonomerranean ,10,11. Ferranean . This sperranean ,14,15,16erranean ,18,19 ans Europe .Pygopleurus israelitus has receptors maximally sensitive to UV, green, and red [P. rhoeas. Considering that color is a major attractant for glaphyrids and bees, we asked whether the geographical spread of P. rhoeas caused modifications of the color of its flowers. To answer this question, we evaluated the spectral reflectance properties of flowers of P. rhoeas in populations in the East and North Mediterranean and Central Europe and modeled flower color appearance, taking into account the spectral sensitivities of the photoreceptors of the glaphyrid beetle, Pygopleurus israelitus (Glaphyridae), and the most common bee pollinator, the honeybee Apis mellifera.Glaphyrids and bees differ substantially with respect to their receptor-based color vision. The glaphyrid beetle  and red , while m and red . These s and red ,22,23, aP. rhoeas is indistinguishable from its European counterpart  color-opponent model and the Maxwell triangle.In the East Mediterranean, populations of phyridae , while i by bees ,15,16. Ged light , most hyed light   and of green foliage (n = 77) in the chromaticity diagrams of the glaphyrid beetle Pygopleurus israelitus and the most common bee pollinator, the honeybee Apis mellifera , at 2 locations in the Northern Mediterranean (Greece), and at 8 different locations in Central Europe (Germany)  .Images of flowers were taken using a digital camera modified for increased UV sensitivity  with a quartz lens . For UV exposures, a narrow bandpass filter was used  that consisted of a Schott UG11 substrate with dielectric coating that totally blocked wavelengths in the visible and infrared ranges while transmitting between 320 and 380 nm with a half-band width of 60 nm. For exposures in the visible spectrum, we used a broad bandpass filter that transmitted light between 400 and 700 nm.The reflectance spectra of flowers and foliage collected in the field were measured between 300 and 700 nm using a spectrometer . A white, diffuse reflectance tile was used as a reference for the measurements. Patches of petals were illuminated by a xenon lamp through an optic fiber while a second optic fiber collected the light reflected by the petals. The sample was illuminated under an angle of 45° to the optical axis of the fiber collecting the reflected light.I(λ) is the illumination spectrum for a flower or leaf with reflectance spectrum S(λ) , middle- (M), and long-wavelength (L)-sensitive photoreceptor k  with spectral sensitivity kR(λ) (Flower colors were plotted in two chromaticity diagrams using the receptor noise-limited (RNL) color-opponent model and the Maxwell triangle. According to the RNL model, colors can be depicted as points in a chromaticity diagram, where the discriminability between colors is given by the Euclidean distance between points c,d. The rum S(λ) , the quaty Rk(λ) a,b is(1kq) was used where quantum catches for stimuli were divided by those for a reference stimulus or background, A coordinate system (kf areAssuming validity of Weber’s law, the color loci can be plotted with the coordinatesωk, were set to Sω = 0.13, Mω = 0.06, and Lω = 0.12 [Note that the axes do not correspond to opponent mechanisms. The Weber fractions or noise values, L = 0.12 . The disThe Maxwell triangle, on the other hand, does not make assumptions about the noise level of photoreceptor mechanisms, representing the most commonly used diagram in studies of trichromatic animals. According to this diagram, receptor coordinates are given byThe location of a point, ightness . To plotNote that these axes are not related to opponent mechanisms. The vertices of the triangle are given by the following coordinates:


Since February 2020, Italy has been seriously affected by the SARS-CoV-2 pandemic. To support the National Health Care system, naso-pharyngeal/oropharyngeal swabs collected from suspected cases of Teramo province, Abruzzo region, are tested at Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise G. Caporale, for the presence of SARS-CoV-2 RNA. Out of 12,446 tested individuals, 605 returned positive results at least once, with prevalence significantly higher in men. A reduction of the level of viral RNA in the first swab per each positive patient collected over time was also observed. Moreover, 81 patients had at least one positive sample and two final negative tests: positivity in swabs lasted from 14 to 63 days, with a median value of 30 days. This shows the potential for the virus to coexist with patients for a long time, although we highlighted intermittent positivity in several cases. The evolution of the SARS-CoV-2 epidemiological situation and knowledge on viral shedding should be closely monitored, to interpret the findings correctly and adjust accordingly the surveillance activities. Eighteen years after the emergence of severe acute respiratory syndrome (SARS) in China, and eight years after the emergence of Middle East respiratory syndrome (MERS) in Saudi Arabia, a novel coronavirus (CoV) epidemic of a probable animal origin, acknowledged as a pandemic on March 11 2020 by the WHO [Severe acute respiratory syndrome-related coronavirus (SARS-rCoV), sub genus Sarbecovirus, genus Betacoronavirus, sub family Orthocoronavirinae, family Coronaviridae, with other known viruses mainly identified in humans, wild carnivores, and bats [The disease, referred to as coronavirus disease 2019 (COVID-19) , is causand bats ,6.The virus was first identified in Wuhan, Hubei province of China, where pneumonia cases of unknown origin were observed since mid-December 2019 , sustainIn Abruzzo, a region located in the central part of Italy, the first case of COVID-19 was recorded in a male patient on 27 February . The patIn order to support the National Health Care system, the Ministry of Health appointed the Istituti Zooprofilattici Sperimentali (IZSs), which are public veterinary institutes, to test the naso-pharyngeal swabs collected from suspected human cases for SARS-CoV-2 . The swaIn addition to a wide overview of the results originating from SARS-CoV-2 diagnostic activities performed at IZSAM in the last 3 months, this paper mainly focuses upon SARS-CoV-2 RNA persistence in naso-pharyngeal/oropharyngeal swabs.The testing of suspected COVID-19 cases and contacts in Teramo province was conducted within the official surveillance programme established by the Italian Health Authorities and did not require ethical approval.Starting from February 29th, naso-pharyngeal/oropharyngeal swabs collected from suspected cases of Teramo province, Abruzzo region, are tested at IZSAM for the presence of SARS-CoV-2 RNA. Samples were collected from the respiratory tract of individuals either hospitalized, or screened as having a contact history with infected individuals, or in the framework of screening programs for health workers belonging to the National Health Care System. ® MTM, Bethesda, MD, USA) in BSL3 bio containment laboratory, starting from a total volume of 200 μL oropharyngeal swab transport medium  or bronchoalveolar lavage (BAL) and nucleic acid purification , according to the manufacturer’s instructions. The second consisted of RNA detection by the TaqManTM 2019-nCoV Assay Kit v1/v2 , whose results are interpreted following the manufacturer’s instructions. Specifically, this test targets three different portions of SARS-CoV-2 genome located in the replicase, the S and the N protein encoding genes, respectively [T values produced for each diagnostic target, we selected the CT value associated with detection of the N protein encoding gene, since it is translated by the most abundant viral subgenomic RNA [Two steps compose the workflow for SARS-CoV-2 RNA detection. The first includes virus inactivation  of the two groups . A two-tailed Mann–Whitney test was applied to compare the two groups: positive/negative and age of each patient. In addition, a Mann–Whitney test was used to assess statistical significance among the CT values for N protein encoding gene. In addition, a two-tailed Mann–Whitney test was applied to compare the median CT values at the first sample and gender. Finally, a Wilcoxon signed rank test was used to assess significance in the trend of CT gene values over time until the first negative swab.Amongst the positive patients, we selected those who returned positive results at least twice, as a cohort, to study the duration of RNA positivity in swabs. A pairwise correlation test was performed to evaluate the correlation between the age of patients, the duration of RNA positivity in swabs, and Cp-value of 0.05.The statistical analysis was performed using StatTools© . The level of significance was set at a From February 29th to May 21th of 2020, IZSAM analyzed a total number of 20,878 naso-pharyngeal swabs collected from 14,200 individuals in Teramo province: 854 swabs tested positive by qPCR  naso-phan = 5,440) and 56.3% females . Moreover, 605 tested positive at least once, and 11,841 were negative. Within the group of positives, 52.2% (n = 316) were male and 47.8% (n = 289/605) were females. The difference observed between genders’ prevalence of infection was statistically significant . Positive patients represented 5.8% of the male group (95% IC 4.9–6.5%) and 4.1% of the female patients (95% IC 3.7–4.6%).Information about gender was available for 12,446 out of 14,200 patients. Taking into consideration these 12,446 patients, 43.7% were males .Information about age was available for 14,016 patients, 626 of which tested positive. The mean age of positive and negative individuals was compared: mean age of negatives was 53.4 years (±20.3 standard deviation), whereas positive individuals were 55.0 years on average (± 20.3 standard deviation). The difference between the mean age of positives and negatives was statistically significant . However, no statistically significant difference was found between April and May   (The C 0.0732) . Moreove= 0.218) .Between March 14th and May 6th, 108 patients tested positive by qPCR more than once. Therefore, they were followed up on until June 3rd for calculation of SARS-CoV-2 RNA persistence in swabs. Up to June 3rd, 81 of the initial 108 patients had a double negative final naso-pharyngeal/oropharyngeal swab, thus indicating a possible recovery from the infection. The difference in days between the date of the first qPCR positivity and the first negative swab in individual patients varied from 14 to 63 days, with a median value of 30 days (± 10.68 days of standard deviation) .T N gene values at the first positive swab and duration of RNA persistence, suggesting higher viral loads were correlated with longer duration of viral shedding   . No corrT N gene in subsequent swabs of the same patient, was significantly higher than the previous one  . Additionally, the difference between genders and CT N gene values at the first positive swab sample was not significant . However, when the correlation test between the CT N gene values at the first positive swab sample and the RNA persistence was performed separately for males and females, a significant correlation was observed only in males   .Of the s −0.349) . Bearing in mind that among the 605 positive patients for which the gender was known, 52.2% were male, we found that the SARS-CoV-2 prevalence in men was significantly higher compared to women. The role of gender in morbidity and mortality of SARS-CoV-2 infection was investigated by Jin et al. , who detDepending on the collected specimen, the timeline of qPCR positivity may vary: qPCR positivity declines slower in sputum, and may still be positive after naso-pharyngeal swabs are negative ,18. ManyT N gene values in positive naso-pharyngeal swabs were compared over time, a reduction of the level of viral RNA was observed in swabs collected in April when compared to those collected in March. In contrast, the absence of a clear statistical difference between May and April was seen , mainly due to the low number of positive samples collected in May (n = 7). A reduction trend of viral RNA levels in naso-pharyngeal swabs was observed across time, which is difficult to explain in light of the lack of consistent viral mutations of potential clinical or pathological relevance [Interestingly, when levels of Celevance  or, alteThe information about the clinical status of all positive persons was not available. Consequently, we can only hypothesize that either the surveillance increased over time, including testing of persons at risk and in significant contact with confirmed cases, or the elimination of infection foci over time was able to reduce the general viral load in the exposure environments . Further investigations are needed to confirm and better explain our findings, including the collection of more data for each positive individual, relevant to the clinical status and the possible way of exposure.T N gene values at the first positive swab sample between males and females was not statistically significant, in contrast to another study in which the virus was identified for a significantly longer period in men than in women [T N gene values at first sampling, seems to indicate a role of gender in the persistence of the infection. It appears that SARS-CoV-2 virus has the potential to coexist with patients for a long time, with serious implications for prolonged required quarantines and global efforts to control the infection [Regarding the duration of viral shedding, a study previously performed in Wuhan reported a median duration of viral shedding in symptomatic patients of 20 days (range 8–37) . In anotin women . Howevernfection . Howevernfection .Two out of the 81 patients had two double negative naso-pharyngeal/oropharyngeal swabs before having one more positive sample, and then finally turning negative (double swabs) again. Respiratory shedding may be intermittent; therefore, a single negative swab could be misleading . A few sLastly, for 27 patients out of the 108 selected as a cohort, it was not possible to estimate SARS-CoV-2 RNA persistence in swabs because of the absence of the double negative swab in our records. A further limitation of this study was the lack of information on the clinical status for each qPCR positive case: we had partial or inconsistent information on the presence or absence of symptoms, therefore an important part of the picture is missing, especially in the interpretation of the cases with conflicting results. For 10 out of these 27 patients, a fluctuation in the outcomes was registered at least once: tests results went from positive to negative and from negative to positive again. The other 17 patients had only positive swabs and never a negative test result in our registry.T N values at the first positive swab are correlated with a longer persistence of qPCR positivity. Those findings suggest a virus coexistence with patients for long time periods, and that a longer recovery time is required when higher viral loads are measured. Nevertheless, to maximize the sensitivity, multiple tests should be performed, if possible, on different collected specimens. We also observed a reduction across the study period of the viral RNA load in naso-pharyngeal swabs, which is difficult to explain with the available information in the registry, but should be better investigated through the systematic and detailed collection of additional data for each positive individual about the clinical status and the possible way of exposure.In our study, SARS-CoV-2 virus persistence in swabs reaches 63 days, with a median value of 30 days (±10.68). Moreover, lower CThe situation on COVID-19 is constantly evolving, and new studies on SARS-CoV-2 are continuously becoming available. As a number of questions remain unanswered, new findings should be actively monitored, with surveillance activities adjusted accordingly.


Diabetes refers to any group of metabolic diseases characterized by high blood sugar and generally thought to be caused by insufficient production of insulin, impaired response to insulin. Globally, patients with type 2 diabetes account for more than 85% of the total diabetic patients, and due to factors, such as obesity, aging, environment and lifestyle, the incidence of diabetes is rising. Salvia miltiorrhiza (SM) is a medicine used to treat diabetes in China. In recent years, it has been reported that SM has the effect of improving type 2 diabetes. However, there is no systematic review of its efficacy and safety yet. Therefore, we propose a systematic review to evaluate the efficacy and safety of SM for T2D.Six databases will be searched: China National Knowledge Infrastructure (CNKI), China Biological Medicine (CBM), China Scientific Journals Database (CSJD), Wanfang database, PubMed, and EMBASE. The information is searched from January 2010 to July 2020. Languages are limited to English and Chinese. The primary outcomes include 2 hour plasma glucose, fasting plasma glucose, hemoglobin A1c, homeostasis model assessment of insulin resistance, and fasting plasma insulin. The secondary outcomes include clinical efficacy and adverse events.This systematic review will evaluate the efficacy and safety of Salvia miltiorrhiza in the treatment of type 2 diabetes.This systematic review provides evidence as to whether Salvia miltiorrhiza is effective and safe for type 2 diabetes.Ethical approval is not necessary as this protocol is only for systematic review and does not involve in privacy data or an animal experiment.INPLASY2020110046. Chronic hyperglycemia and insulin resistance can lead to chronic damage and dysfunction of various tissues, which seriously affects peoples quality of life. Long-term chronic damage and dysfunction are the main causes of death from diabetes. However, there are currently limited options of the drugs and treatment for diabetes, people are embarking on looking for alternative medicines from traditional herbal medicines.Diabetes refers to any group of metabolic diseases characterized by high blood sugar and generally thought to be caused by insufficient production of insulin, impaired response to insulin. Globally, patients with type 2 diabetes account for more than 85% of the total diabetic patients,–7 Many clinical trials have found that SM has a significant improvement effect on type 2 diabetes.–10Salvia miltiorrhiza (SM) is a medicinal plant with a history of more than 2000 years of use in China. Traditional Chinese Medicine (TCM) uses SM to treat palpitation, stroke, perimenopausal syndrome, anemia and other diseases.Unfortunately, there is currently no systematic review of the safety and effectiveness of SM in the treatment of T2D. Therefore, we propose a protocol for a systematic review to evaluate the effectiveness and safety of SM in the treatment of T2D patients, so as to provide a rigorous evaluation of the existing evidence.22.1https://inplasy.com/) with number INPLASY2020110046. Ethical approval is unnecessary because this study only involves the data of published previous studies.This protocol of systematic and meta-analysis review has been registered on Inplasy  can be included. Observation studies, animal research, case report, review, and meta-analysis are excluded.2.2.2 These types of patients will not be included: patients with acute complications of diabetes; patients with severe heart disease, liver and kidney dysfunction, mental illness, or a relevant drug allergic history and patients during pregnancy or lactation.Patients who have been diagnosed with T2D will follow the American Diabetes Guidelines. There are no restrictions on gender, age, course of the disease, TCM syndrome, and race.2.2.3Analyzed interventions included Salvia miltiorrhiza used as monotherapy, Chinese herbal compound prescription and related alternative therapies.2.2.4Patients diagnosed with type 2 diabetes but who have not received it or who have only received nonpharmacological treatment.2.2.5The primary outcomes include1.2 hour plasma glucose2.Fasting plasma glucose3.Hemoglobin4.Homeostasis model assessment of insulin resistance5.Fasting plasma insulin and adverse events.2.3We search the following databases from January 2010 to July 1, 2020: China National Knowledge Infrastructure, China Biological Medicine, China Scientific Journals Database, Wanfang database, PubMed, EMBASE. We will search English and Chinese articles for review, and collect additional references from review references and original research articles.2.4Two review authors will search the literature independently with cross- check. Any inconsistency will be solved by a third reviewer. Manual search will be performed if relevant literatures are found in the included studies. The electronic search will be conducted using a combination of following keywords diabetes, T2D, type 2 diabetes, Non-Insulin-Dependent diabetes mellitus, Chinese patent medicine, Chinese herbal drugs, herbal, salvia miltiorrhiza, salvia, dan shen, randomized controlled trial, controlled clinical trial, trial, RCT, randomized, randomly. The search strategy for PubMed is presented in Table 2.52.5.1Two reviewers will perform literature screening, study selection, and data extraction independently. The literature obtained will be imported into EndnoteX9 to screen the title and abstract, the duplication and studies failing to meet the pre-specified inclusion criteria will be excluded. After reading the full text of the remained literature and discussing within the group, the final included studies will be determined. The corresponding author of original RCT will be contacted when the full text is unavailable. Disagreements will be solved by consulting a third-party arbitrator or discussing within a group. The flowchart of studies searching process is shown in Figure 2.5.2For each RCT, we extracted the following information:1.general information, including name of the first author and year of publication;2.participant characteristics, including sample size, T2D severity, gender composition, mean age, and diagnostic criteria;3.intervention details, including the experimental medicine and control group care, doses of medications, duration of treatment; and4.outcome measures and intergroup differences.Extract data from the primary and secondary results for further evaluation. The inconsistency between the 2 reviewers will be resolved by the third reviewer.2.5.3The quality of the studies will be assessed by using the Cochrane Handbook 5.1.0 (Cochrane Handbook 5.1.0). The assessment will include random sequence generation, randomization correctness, allocation scheme hiding, blinding of patients and implementers, accuracy of data results, and other risk of bias. The risk of low bias is expressed as “low risk” and the risk of high bias is expressed as “high risk”. The information provided in the studies is inaccurate or does not provide sufficient information for the bias assessment to be expressed as “unclear risk”. The above content evaluation was independently evaluated by 2 researchers, and any differences will be resolved through discussions with the third reviewer.2.5.4Two reviewers will analyze the data independently using RevMan 5.3.5 Risk ratio with 95% confidence interval will be adopted for the dichotomous data, whereas the mean difference or standardized mean difference with 95% CI will be utilized for the continuous data.3I2 and Q tests will be applied to evaluate the heterogeneity with the cut-off value of I2 = 50. If I2 > 50% and/or Q test <0.10, the heterogeneity will be deemed significant.Assessment of reporting biases. A funnel plot will be performed to assess any publication bias when more than 10 RCTs are included. In additional, Egger regression and Begg correlation test will also be performed to identify the funnel plot asymmetry. The Cochrane 4I2 < 50, and a random-effect model will be employed if I2 ≥ 50. Subgroup analysis or meta-regression will be performed to assess the potential sources and present reasonable explanations for the heterogeneity. Sensitivity analysis will be applied to evaluate the stability of the pooled results of included RCTs according to the methodological quality, sample size and missing data.In line with the Cochrane guideline, a fixed-effect model will be utilized to pool and analyze the outcome data if 5The Grading of Recommendations Assessment, Development and Evaluation (GRADE) guidelines will be utilized to grade the quality of evidence as very low, low, moderate, or high.6 On the other hand, high blood sugar often leads to a series of complications, and severe cases can even cause death.,14 At present, our understanding of the pathogenesis of diabetes is not fully understood, and the drugs commonly used in clinical treatment of type 2 diabetes are often accompanied by some side effects, such as hypoglycemia, liver damage and gastrointestinal discomfort, etc,16. Therefore, people urgently need some supplementary and alternative medicines to improve the treatment of diabetes.Diabetes has become a major public health problem worldwide, especially in developing countries.,18 As a natural medicine that has been used in China for many years, SM has many advantages that synthetic medicines do not have. More and more evidence shows that Danshen can lower blood sugar and improve diabetes complications.–21SM was often used with other herbs in ancient China to treat various diseases in TCM clinical practice, including diabetes (consumptive thirst in TCM). With modern pharmacology in-depth research on the components and effects of SM, an increasing number of people attach importance to SM prevention and treatment of diabetes.However, currently no systematic review and meta-analysis have been conducted regarding the efficacy and safety of SM for the treatment of patient with T2D. The findings of this study may yield helpful evidence for the clinicians and investigators concerned in decision-making process about the efficacy and safety of SM for patients with T2DThis study was supported by The Project of Science and Technology Department of Sichuan Province for Youth Fund (2020JDRC0115). The authors would like to thank Sanyin Zhang and Zhipeng Wu for critically reviewing the manuscript.Data collection: Ying Guo, Jianfeng Sun.Statistical analysis: Renyan Zhang, Jianfeng Sun.Supervision: SanYin Zhang, Zhipeng Wu.Writing – original draft: Ying Guo, Peng Yang.Writing – review & editing: Sanyin Zhang, Zhipeng Wu.