Though radiotherapy has been widely used for knee pigmented villonodular synovitis (PVNS), there is few literatures about radiotherapy for the treatment of PVNS hip. Thus, the purpose of this study was to analyze the clinical outcomes of endoscopic synovectomy with/without radiotherapy postoperatively of PVNS hip.We performed a retrospective study of patients who underwent endoscopy in our hospital from November 2010 to January 2021. Inclusion criteria was patients with magnetic resonance image (MRI) signs, endoscopic findings and/or histological evidence of PVNS. Exclusion criteria was patients lost follow-up. All patients underwent synovectomy endoscopically and were divided into two groups depending on receiving postoperative radiotherapy or not. The primary outcome measurements were the recurrence of PVNS, receiving revision, and/or converting to total hip arthroplasty (THA). The secondary outcome measurements were the patient-reported outcome (PRO) collected at pre- and post-operation, which consist of Hip Outcome Score Activities of Daily Living (HOS-ADL), modified Harris Hip Score (mHHS), International Hip Outcome Tool-12 (IHOT-12), Non-arthritic Hip Scale (NAHS), and visual analog scale (VAS).In a case series of 16 patients , 4 (25%) cases were localized type and 12 (75%) cases were diffuse type. The average follow-up was 44.8 ± 38.2 months . 8 (50%) cases  received radiotherapy postoperatively, and the rest  received endoscopic treatment alone. At the latest follow-up, 3 (18.75%) cases  who did not receive radiotherapy converted to arthroplasty. The preoperative HOS-ADL, mHHS, IHOT-12, NAHS, VAS scores of remaining 13 patients were 63.1 ± 19.1 , 54.8 ± 20.1 , 50.9 ± 15.4 ,51.6 ± 15.9 , 6.0 ± 1.4  points, respectively. The latest HOS-ADL, mHHS, IHOT-12, NAHS, VAS scores of the 13 patients were 79.7 ± 10.8 , 78.6 ± 9.1 , 74.7 ± 9.7 , 78.9 ± 18.7 , 3.1 ± 1.2  points respectively.Endoscopic synovectomy can achieve satisfactory PRO in PVNS hip patients. Besides, postoperative adjuvant radiotherapy can achieve higher hip survivability than synovectomy alone in this present study. Pigmented villonodular synovitis (PVNS) is a rare, benign disease that is characterized by destructive and recurrent. This disease was first documented by Chassaignac . in 1852PVNS has no specific symptoms, usually manifesting hemorrhagic effusion, swelling, pain, and consequent movement restriction, which might be misdiagnosed. Nowadays, the development of magnetic resonance imaging (MRI) has become an effective and instructive method for diagnosing PVNS. PVNS can be divided into two types , 9: diffTo our best knowledge, this is the first case series study that aim to analyze the clinical outcomes of endoscopic synovectomy with/without radiotherapy postoperatively of PVNS hip.This study was a retrospective case series in which all patients underwent arthroscopy at our hospital from November 2010 to February 2021.Inclusion criteria was patients with MRI signs, endoscopic findings, and/or histological evidence of PVNS  who underwent endoscopy at our hospital. Among those patients, 1 case was misdiagnosed as inflammation, 1 case as rheumatoid arthritis, 1 case as osteonecrosis of the femoral head, 2 cases as synovial chondromatosis, in other medical facilities previously. There were 4 (25%) cases of localized type and 12 (75%) cases of diffuse type. All patients underwent synovectomy endoscopically, among which 8  patients received radiotherapy postoperatively. For the femoral head cartilage injury, 5 cases were degree IV, 4 cases were degree III, 4 cases were degree II, and 3 cases were degree I. And for the acetabular cartilage injury, 5 cases were degree IV, 1 case was degree III, 1 case was degree II, 9 cases had no cartilage injury. All injuries were located at anterosuperior sites of femoral head or acetabulum.2 . The mean follow-up was 44.8 months . The symptom duration time before the operation was 16.9 months   or not (NRT group). Due to long interval after surgery (24 months) of a patient, the patient was not included in the RT group for statistical analysis. At the latest follow-up, no recurrence or revision were in those patients, but 3 cases  of NRT group converted to THA , 54.8 ± 20.1 , 50.9 ± 15.4 ,51.6 ± 15.9 , 6.0 ± 1.4  points, respectively. The latest HOS-ADL, mHHS, IHOT-12, NAHS, VAS scores of the 13 patients were 79.7 ± 10.8 , 78.6 ± 9.1 , 74.7 ± 9.7 , 78.9 ± 18.7 , 3.1 ± 1.2  points, respectively. , intensity modulated radiotherapy (IMRT), and other novel radiotherapy technologies. Those emerging technologies have the advantages of precise positioning and accurate design.Radiotherapy has been used widely for refractory cases in the knee joint, but there are few cases in the setting of radiotherapy of hip PVNS exist in the contemporary literature. To our best knowledge, this is the first study aimed to compare the clinical outcomes of hip PVNS patients who underwent CRT or IGRT followed by synovectomy endoscopically with those who received isolated synovectomy. In this study, we found a higher rate of hip joint survivability of the RT group than NRT group, which might provide the evidence that adjuvant radiotherapy treatment after endoscopic synovectomy for hip PVNS can be an effective and safe method.There are several important limitations of this study. First, though the size of the sample is relatively large for known reports of a single center, the total sample size was still small, and there is a bias in patients’ choice of radiotherapy, thus more patients were needed to compare the results. Second, though satisfactory average mid-term PRO gained in our study, the long-term outcome remains to be seen. Third, the time span of this study was more than 11 years, which may affect the outcomes because of the improvement of surgery and radiotherapy technology. Fourth, treatment for concomitant pathology were performed, which made it difficult to distinguish whether the improved PRO was due to the treatment of PVNS or other hip lesions.Endoscopic synovectomy can achieve satisfactory PRO. Besides, postoperative adjuvant radiotherapy can achieve higher hip survivability than synovectomy alone in this present study.


Despite the fact that fat-tailed sheep raised for meat production are well known for being resilient to harsh environmental conditions such as pasture scarcity or low-quality feedstuffs, no studies regarding feed restriction have been performed on fat-tailed dairy sheep. In this study, prepartum feed restriction from week −5 to week −1 relative to parturition did not affect body weight. Similarly, postpartum feed restriction from week 1 to week 5 relative to parturition did not affect body weight. However, both prepartum and postpartum feed restrictions affected blood metabolites and hormones, which decreased both colostrum and milk yields postpartum.n = 10) or the feed-restricted  groups from week −5 to week 5 relative to parturition. Despite dry matter intake being decreased in the FR group compared to the Ctrl throughout both prepartum and postpartum periods, no differences in BW were detected between groups in any of the studied periods. Feed restriction increased both free fatty acids and beta-hydroxybutyrate concentrations during both prepartum and postpartum periods. Similarly, feed restriction increased triglyceride concentration postpartum. Additionally, feed restriction increased insulin and growth hormone and decreased prolactin concentrations during both prepartum and postpartum periods. Feed restriction caused a decreased colostrum yield and a relative increase of the main colostrum components in the FR group. Similarly, milk yield decreased in the FR group compared to the Ctrl group, although milk components were not affected. In conclusion, feed restriction did not affect BW but decreased colostrum and milk yield in fat-tailed dairy sheep.This study aimed to investigate the effect of prepartum and postpartum feed restriction on body weight (BW), blood metabolites, and hormones as well as colostrum and milk yields and compositions in fat-tailed dairy sheep. In this study, 20 multiparous and pregnant ewes were randomly allocated to either the control (Ctrl;  In developing countries, livestock systems located in tropical and sub-tropical regions are heavily dependent on natural resources . In these countries, decreased pasture availability and quality during the dry season have important consequences on the performance and health of dairy ruminants , especiaFat-tailed sheep are raised in semi-arid regions of Eastern and Southern Africa, Central Asia, and numerous countries in the Middle East . The comBased on the above-mentioned facts, the current study aimed to investigate the consequences of pre- and postpartum feed restriction on body weight (BW), blood metabolites, and hormones as well as colostrum and milk yield and composition in dairy fat-tailed ewes. This study hypothesizes that feed restriction around parturition affects BW, blood metabolites, and hormones as well as colostrum and milk yield and composition in fat-tailed sheep.The present study was performed at the experimental farm of Yasouj University . All animal procedures followed the ethical law on Animal Protection and were approved by the Committee of Animal Experiments  under the procedure 950441007-13845. During the entire experiment, all ewes were visually healthy and had no signs of diarrhea.n = 10; Turkey-Qashqai, n = 10) with an average age 40.8 ± 6.2 months and BW 56 ± 1.8 kg, which provided a power analysis = 0.85. The experimental period lasted from week −5 to week 5 relative to parturition. During the trial, all animals were kept in individual pens (1.2 × 1.0 m) located in a closed barn. Each pen was equipped with individual drink and feed containers. Animals were placed outdoor for sunlight, exercise, and hygiene operations three h twice a week after morning feeding. Three days before the expected parturition, ewes were transferred to a parturition pen (3.0 × 2.0 m) with clean straw bedding and free access to water and feed. After the first week postpartum, animals returned to the individual pens (1.2 × 1.0 m), where they stayed until the end of the experimental period .This experiment used 20 multiparous and pregnant fat-tailed dairy ewes  and the feed-restricted  groups. From week −5 relative to parturition, ewes from the Ctrl group received the dry diet. Ewes from the FR group were fed with different energy levels from week −5 to week −1 relative to expected parturition. Thus, the FR group was fed with a diet equivalent to 100, 50, 65, 80, and 100% of the energy content of the dry diet at weeks −5, −4, −3, −2, and −1 relatives to expected parturition, respectively. After parturition, ewes from the Ctrl group were fed with a TMR diet formulated to fulfill 100% of the energy requirements recommended by the NRC [From week −7 to week −5, all animals were fed with a total mixed ration (TMR) diet formulated to fulfill 100% of the energy requirements recommended by the National Research Council [The individual feed intake was recorded daily by weighing the offered TMR and the residual TMR of the next morning before feeding. Feed samples were collected to determine the DM of the diets and calculate dry matter intake (DMI). The dry matter (DM) content of the diets was determined according to the method described by the Association of Official Analytical Chemists . IndiviColostrum samples (50 mL) were collected immediately at parturition. In addition, milk samples (50 mL) were collected at 0800 and 1600 twice a week during the experimental period. Both colostrum and milk samples were treated with Bronopol tablets  and then stored at −20 °C.g for 20 min at 4 °C . Then, the plasma was aliquoted (1.5 mL) and stored at −20 °C.During the entire experimental period (from week −5 to week 5), blood samples were collected weekly from the jugular vein using heparinized vacuum tubes (6 mL) at 0730. All blood samples were kept in wet ice and then centrifuged at 3000× Colostrum and milk samples were analyzed for fat, protein, lactose, solids not fat (SNF) content, and density using a Lactoscan milk analyzer . Fat-corrected milk  as well as milk energy content  were calculated based on the following equations as described by Milis , and theThe plasma concentration of glucose (#1500017), triglycerides , cholesterol (#1500010), total protein , albumin (#101500), urea (#1400029), creatinine (#1400009) as well as the plasma activity of lactate dehydrogenase , alanine aminotransferase , and aspartate transaminase  were determined using an automatic chemical analyzer and commercial kits . The intra-assay coefficients of variation were 1.5, 1.6, 0.9, 0.9, 1.3, 3.3, 3.2, 2.5, 3.6, and 2.4%, respectively. The inter-assay coefficients of variation were 0.9, 1.2, 1.1, 1.3, 1.5, 4.1, 1.8, 1.7, 1.9, and 2.2%, respectively.Plasma free fatty acids  and beta-hydroxybutyrate  concentrations were measured using commercial kits  following the manufacturer’s instructions. The intra-assay coefficients of variation were 4.4 and 5.1%, respectively. The inter-assay coefficients of variation were 4.7 and 37%, respectively. Insulin (# CSB-E17044Sh), growth hormone , prolactin (# CSB-E13161Sh), and progesterone (#CSB-E13176Sh) concentrations were determined using commercial ELISA kits  following the manufacturer’s instructions. All ELISA kits used in this study were validated for being used in sheep plasma. The intra-assay coefficients of variation were 6.7, 5.6, 5.3, and 4.9%, respectively. The inter-assay coefficients of variation were 12.6, 15.2, 14.7, and 12.2%, respectively.p ≤ 0.05. Results are presented as least squares means (LSM) ± standard error of the mean (SEM).The data were tested for normal distribution using the UNIVARIATE procedure of SAS . Two datasets were created based on week relative to parturition. The prepartum dataset included all data collected from week −5 to week −1. Similarly, the postpartum dataset included all data collected from week 1 to week 5. Both datasets were evaluated using the MIXED procedure of SAS. The model included feed restriction (Ctrl and FR), time (either from week −5 to week −1 or from week 1 to week 5), and the interaction (feed restriction × time) as fixed effects. Breed (Lori-Bakhtiari and Turkey-Qashqai) was set as a random effect, and the individual ewe was set as a repeated subject. Significant effects were considered when p > 0.05). From week 1 to week 5, all animals  were visually healthy.In the present study, none of the animals showed dystocia at parturition and did not show symptoms related to metritis and mastitis during the entire experimental period. Pregnancy length was 151 ± 1.13 and 152 ± 1.12 days in the Ctrl and FR group, respectively (data shown as mean ± standard deviation). In this study, all ewes gave birth to a single lamb. No differences in birth BW were detected between singleton lambs born from the FR group and those born from the Ctrl group . The induced feed restriction decreased DMI in the FR group compared to the Ctrl group during both prepartum and postpartum periods (p ≤ 0.05). As expected, no differences in DMI were detected between the Ctrl and FR groups at week −5 relative to parturition (p > 0.05). However, DMI was lower in the FR group than in the Ctrl group at weeks −4, −3, −2, and −1 relative to parturition (p ≤ 0.05). During the postpartum period, no differences in DMI were observed between groups at week 1 and week 5 postpartum (p > 0.05). However, DMI was lower in the FR group than in the Ctrl group at weeks 2, 3, and 4 postpartum (p ≤ 0.05).As shown in p > 0.05). After parturition, BW decreased continuously in both the Ctrl and FR groups until the end of the postpartum period (p ≤ 0.05), although no differences were detected between groups (p > 0.05).As observed in p > 0.05).p ≤ 0.05) and time (p ≤ 0.05). In both periods, FFA concentrations were higher in the FR group than in the Ctrl group (p ≤ 0.05). During the prepartum period, both groups had increased FFA concentrations (p ≤ 0.05). During the postpartum period, FFA concentrations remained constant in the Ctrl group (p > 0.05). In contrast, the FR group had decreased FFA concentrations from week 1 to week 3, which was followed by a slight increase at week 4 and a decrease at week 5 (p ≤ 0.05).During both prepartum and postpartum periods, FFA concentrations A were afp ≤ 0.05) and time (p ≤ 0.05). During the prepartum period, BHB concentrations were higher in the FR group than in the Ctrl group (p ≤ 0.05). Both groups had increased BHB concentrations from week −5 to week −1 during the prepartum period (p ≤ 0.05). In the postpartum period, BHB concentrations were higher in the FR group than the Ctrl group (p ≤ 0.05). During the postpartum period, BHB concentrations remained constant in the Ctrl group (p > 0.05). In contrast, the FR group had decreased BHB concentrations from week 1 to week 2 followed by a slight increase from week 2 to week 4 when FFA concentrations decreased again until week 5 (p ≤ 0.05).During both prepartum and postpartum periods, BHB concentrations B were afp > 0.05) or time (p > 0.05). During the postpartum period, TG concentrations were affected by feed restriction (p ≤ 0.05) and time (p ≤ 0.05). Triglyceride concentrations were higher in the FR group than in the Ctrl group (p ≤ 0.05). In both groups, TG concentrations remained constant from week 1 to week 4 and increased in week 5 (p ≤ 0.05).During the prepartum period, TC concentrations C were nop ≤ 0.05). During the prepartum period, insulin concentrations were lower in the FR group compared to the Ctrl group in week −2 (p ≤ 0.05). No differences were detected between groups during the rest of the prepartum period (p > 0.05). During the postpartum period, the FR group showed lower insulin concentrations than the Ctrl group at weeks 3 and 4 (p ≤ 0.05). No differences between groups were detected at weeks 1, 2, and 5 (p > 0.05).p ≤ 0.05). During the prepartum period, no differences between groups were detected at week −5 (p > 0.05). However, the FR group showed higher GH concentrations than the Ctrl group during the rest of the prepartum period (p ≤ 0.05). During the postpartum period, no differences were detected between groups at weeks 1, 2, and 3 (p > 0.05). However, the FR group showed higher GH concentrations than the Ctrl group at weeks 4 and 5 relative to parturition (p ≤ 0.05).An interaction between feed restriction and time was detected for GH concentrations B during p ≤ 0.05). During the prepartum period, no differences were detected between groups at weeks −5 and −1 (p > 0.05). However, the FR group showed lower prolactin concentrations than the Ctrl group at weeks −4, −3, and −2 (p ≤ 0.05). During the postpartum period, prolactin concentrations were similar between groups at week 1 (p > 0.05). However, the FR group showed lower prolactin concentrations than the Ctrl group during the rest of the postpartum period (p > 0.05).An interaction between feed restriction and time was detected for prolactin concentrations C during p ≤ 0.05). During that period, progesterone concentrations decreased progressively from week −5  to week −1  (p ≤ 0.05). During the postpartum period, progesterone concentrations were not affected by either feed restriction (p > 0.05) or time (p > 0.05).Progesterone concentrations were only affected by the time during the prepartum period (p ≤ 0.05). Fat, lactose, protein, and SNF percentages in colostrum were higher in the FR group than in the Ctrl group (p ≤ 0.05).As described in p ≤ 0.05; p ≤ 0.05). In the FR group, milk yield decreased from week 1 to week 3 (p ≤ 0.05), while milk yield increased from week 3 to week 5 (p ≤ 0.05). Fat, lactose, and protein percentages and SNF and EVL were not affected by either feed restriction or time postpartum (p > 0.05) in both the Ctrl and FR groups.Milk yield was lower in the FR group compared to the Ctrl group until the end of the experimental period  and hormones , which in turn decreases colostrum and milk yields postpartum.


Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between  The resistive pulse method (RPM) is used for evaluating the transient ionic current blockade associated with the translocation of individual nano- to microsized particles passing through an appropriate diameter pore. Moreover, it is applicable in proving small objects by using pulse-like electrical signals. In addition, because the measured ionic current blockade signals possess information regarding the properties of these particles such as size , shape ,3,4, surSaccharomyces is well-known as the key yeast species in beer fermentation, and S. pastorianus is used in bottom-fermented beer  and was placed in the middle of the model (z = 0 μm).To elucidate the characteristic ionic current waveforms originating from the morphological features of budding yeasts passing through a pore, numerical simulations based on finite element methods were conducted ,4. The gLx) was positioned along the z-axis. The multiphysics simulation was conducted by simultaneously solving Equation (1) the continuity equation at a steady-state (j is the current density) for the applied electric potential of Vc, Equation (2) the Poisson–Boltzmann equation for the electrostatic potential of Vs, Equation (3) the Nernst–Planck equation for the i ion concentration of ci, and Equation (4) the incompressible Navier–Stokes equation for the hydrodynamic pressure and the flow field of p and U:A yeast model [black arrow in Figure 4a] of a given size , a surface charge of −0.015 C/m2, estimated from the zeta potential by using the Grahame equation [z = ±50 μm and r = 50 μm were assumed to be ci = 2.6 mM. This ci value was estimated based on the assumption that the total ionic current was 100 nA, which corresponded to the experimentally measured baseline current. The other boundary conditions were similar to those in a previous study [The boundary conditions for Equation (1) [equation ,25, was Vc + Vs) distribution is shown in Figure 4a. The total ionic current I was calculated by the surface integral for j in the z-direction (jz) at z = 50 μm over the r–θ surface, expressed asThe resulting electric potential (σw = (18.2 MΩ·cm)−1, F = 9.649 × 104 C/mol, e = 1.602 × 10−19 C, kB = 1.381 × 10−23 J/K, T = 293.15 K, εw = 7.097 × 10−10 F/m, and η = 1.002 × 10−3 Ns/m2 [All numerical simulations were performed with COMSOL Multiphysics 5.0  using the physical parameters of S. pastorianus cells were round to ovoid shapes with a size of 5.6 ± 0.9 × 6.9 ± 1.2 μm [D. anomala and D. bruxellensis were spindle-shaped at 2.9 ± 0.8 × 13.6 ± 5.7 μm and 2.5 ± 0.5 × 7.9 ± 3.2 μm, respectively [S. pastorianus were clearly different from those of D. anomala and D. bruxellensis. Budding was observed in some cells [Light microscopic images of cells are shown in ± 1.2 μm a. Both Dectively b,c. The me cells a–c.Vb = 0.1 V between the cathode-side and the anode-side microchambers, and the baseline had a current level of approximately 100 nA. The electrical conductivity (σ) was determined to be 0.104 ± 0.06 S/m. The electric field generated by the bias voltage yields electric forces that drive the cells and translate them through the pores. As illustrated in We applied a bias voltage of S. pastorianus, D. anomala, and D. bruxellensis, respectively. The size and deformability of the sensed particles were estimated from the Ip values generated by the RPM [Ip values of S. pastorianus, D. anomala, and D. bruxellensis were 6.16 ± 3.96 nA, 0.99 ± 0.58 nA, and 0.75 ± 0.46 nA, respectively, which are significantly different. td can be utilized to elucidate the surface charge of the sensed particles [S. pastorianus, D. anomala, and D. bruxellensis were −28.99 ± 0.50 mV, −16.50 ± 0.64 mV, and −14.42 ± 0.90 mV, respectively. Contrary to the prediction from the results of zeta potential measurement, the td values were 1.31 ± 0.89 ms, 1.07 ± 1.12 ms, and 0.93 ± 0.90 ms with statistical differences between S. pastorianus, D. anomala, and D. bruxellensis, respectively. Since the Ip values for Dekkera spp. are extremely smaller than that of S. pastorianus, it is considered that Dekkera spp. pass through the pore with a longitudinal direction. The minor axis of Dekkera spp. is less than half that of S. pastorianus, and the spindle-shaped Dekkera spp. are thought to be more mobile than the round shape of S. pastorianus in solution, so it may be faster to pass the pore [td values indicating the residence time in the pore becomes shorter in long Dekkera spp. compared with short S. pastorianus. the RPM ,6,7. Thethe pore . TherefoIp and td measurements by the RPM for these yeasts was confirmed by three different experiments [Ip on log, td on log, and scatter plot of Ip–td on log-log, and the BDs for each cell are shown in D. bruxellensis, D. anomala, and S. pastorianus, respectively. The electrical current records and Ip-td scatter plot by RPM of a mixture of two types of yeast from S. pastorianus, D. anomala, and D. bruxellensis are shown in S. pastorianus and D. anomala [S. pastorianus and D. bruxellensis [D. anomala and D. bruxellensis [Ip-td scatter plot by RPM analysis of cell mixture and single measurement [S. pastorianus from Dekkera spp. can be expected.The reproducibility of eriments  [Figure eriments a. The hi anomala a, and S.ellensis b. Relatiellensis c. The cesurement b is veryS. pastorianus, D. anomala, and D. bruxellensis were estimated on log10Ip to be 92.4 ± 1.9%, 54.5 ± 5.3%, and 62.6 ± 3.1%, respectively [S. pastorianus was detected with high sensitivity. On the other hand, recall rates of S. pastorianus, D. anomala, and D. bruxellensis were estimated to be 56.8 ± 8.9%, 19.5 ± 13.2%, and 66.8 ± 5.4% on log10td, respectively. Highly sensitive detection of S. pastorianus was not observed. By performing discriminant analysis on the log10Ip–log10td plane, a significant improvement in the recall rate for S. pastorianus was 96.3 ± 0.8% compared to log10Ip and log10td . The limit of detection was 2.4%, which gave a signal at 3SDs above the backgrounds. The accuracy of S. pastorianus identification by PCR is reported to be 94.6% [Ip–td analysis by RPM for the quantitative detection of S. pastorianus in solution and the accurate detection of Dekkera spp. contamination.Recall rates of ectively . S. pastbe 94.6% . RPM canS. pastorianus, 87.6% of the waveforms [S. pastorianus on microscopic images (data not shown), which is close to the appearance rate of the shoulder-shaped waveform in the RPM.In the RPM analysis for aveforms b, blue waveforms b, cyan oaveforms b, purpleLx for the yeast model is a variable that defines the distribution of Vc, Vs, ci, p, and U, including j and ρ in Equations (1)–(4), and affect I in Equation (5) [La = 6.9 μm on the major axis and Lb = 5.6 μm on the minor axis represents a nonbudding S. pastorianus (i). The budded S. pastorianus (cyan and purple) was modeled by merging a bud (daughter cell) to the forward and backward of the mother cell (ii and iii). The sizes of the mother cell (La and Lb) and those of daughter cells  were assumed from the microscopic images. I during the yeast z-position displacement of zyst = 40 to −40 μm. The peak of the current blockade appeared at zyst = 0 μm [La and Lb at zyst = 0 μm [zyst > 0 for  and zyst < 0 for . These corresponded to the passage of the budded S. pastorianus model through the pore in the order of daughter–parent  or parent-daughter , and the appearance of the shoulder was related to the daughter cell size. The simulated waveforms [S. pastorianus can be quantitatively detected by measuring the shouldered waveform of the current ionic blockade. The contributions of the size and orientation of yeasts to the waveform can explain the RPM for the investigated Dekkera spp., in which clear shoulders were not seen. Dekkera spp. has an elongated spindle shape, and the major axis of D. anomala is larger than the pore diameter [Dekkera spp. is apparently smaller than that of S. pastorianus [Ip values obtained are smaller than those of S. pastorianus [Dekkera spp. may be insufficient by the RPM analysis employed in this study. A smaller-sized pore is necessary to improve the resolution for analyzing the budding of Dekkera spp. These results indicate the potential application of RPM for the monitoring of growth viability and fermentation of yeasts by the quantitative detection of budding cells [We simulated whether the shoulder shape of the waveform shows budding by using multiphysics simulations. The size parameter tion (5) ,29. In Ft = 0 μm d, i–iii.t = 0 μm c, i–iii.aveforms d were inaveforms b. Buddindiameter b,c. Thistorianus a–c, theitorianus e–g. The ng cells ,31,32.S. pastorianus from Dekkera spp. by measuring multiple parameters such as cell size, and the quantitative detection of budding S. pastorianus by evaluating the shape of the waveform of the current ionic blockade. We demonstrated the proof-of-concept of RPM for the detection of Dekkera spp. contamination in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.In this study, we demonstrated the potential of RPM analysis using a micropore device with a 10-μm diameter pore for accurate discrimination of


The negative associations were found for NO2 per 10 μg/m3 increase from lag 15 to 19 day. The high PM2.5 concentration (135 μg/m3) was significantly associated with the increased risk of varicella . For NO2, the negative association was found at high concentration (75 μg/m3) on lag 15 to 20 day; and the positive relationship was shown at low concentration (10 μg/m3) on lag 15 to 20 day. Exposure to PM2.5 and NO2 were significantly associated with the risk of varicella among adults. Little attention has been paid to the relationship between air pollutants and varicella among adults. We used data collected in Qingdao, China from 2014 to 2019. A combination of quasi-Poisson generalized linear model (GLM) and distributed lag non-linear model (DLNM) was applied to evaluate the association between exposure to air pollutants and varicella. And the effects of exposure to extremely high concentration (at 97.5th percentile) and low concentration (at 2.5th percentile) of air pollutants on varicella were also calculated. The level II of GB3095-2012 was used as the reference. A 10 μg/m In China, the average annual reported incidence rate of varicella in the past 2016–2019 was 55.05/100,000, which was much higher than that in 2005–2015 3. The incidence rate of varicella increased year after year during 2016–2019 , and so is it in adults4.Varicella  is an airborne disease caused by the varicella-zoster virus (VZV)6. In adults, the incidence rate of varicella is considerably lower than that in children. Even though the incidence rate of varicella in adults is not high, severe complications including pneumonia, encephalitis, and even death can occur among adolescent and adult patients, especially in the first infection in adulthood8. In Qingdao, varicella vaccine for target age children is included in the expanded immunization program. Compared with children, varicella vaccine for adults is outside the scope of immunization program. Therefore, Varicella is still considered a critical public health issue worldwide, with about 4.2 million severe varicella complications and approximately 4200 related deaths per year estimated by the World Health Organization (WHO) in 20149.Varicella is a common acute infectious disease in childhood, and is generally benign and self-limiting10. It have reported that exposing to air pollutants could adversely affect some chronic diseases13 and infectious diseases such as tuberculosis14, mumps15, hand-foot-mouth disease16, measles17, etc. Air pollution was proven to make humans more susceptible to the invasion of pathogens, then increasing the chance of the infection of respiratory virus18. For this reason, air pollution may also be related to the incidence of varicella.Ambient pollution has become a serious global concern for its harm to human health10 and varicella. Only one study conducted by Yu et al. found that high PM10 was associated with the increase risk of varicella19. It is believed that other particulate matter such as PM2.5 and NO2 (nitrogen dioxide) are the important exposures to infectious diseases21. Besides, the study focusing on adult varicella was scarce. Due to adult varicella is often more serious, it is particularly important to study the varicella among adults. In the present study, we investigated the relationship between PM10, PM2.5, NO2, SO2 (sulfur dioxide), O3 (ozone) and the incidence of varicella among adults (≥ 20 years old) in Qingdao, eastern China during 2014–2019.To our best knowledge, there is no report about the association between other air pollutants expect PMQingdao 36°04′N 120°23′E) is located between longitude 119°30'E to 121°E and latitude 35°35'N to 37°09'N in Shandong Province, China . Generally, varicella can be diagnosed by doctors according to fever and characteristic rash. In Qingdao, medical institutions must report varicella cases to local Center for Disease Control and Prevention (CDC) through NDSS within 24 h after diagnosis and complete case review.10, PM2.5, NO2, SO2 and maximum 8-h moving average ozone concentration (O3-8 h) of nine sites were used as the exposures in our analysis.The daily (24-h) pollutant data were collected from Qingdao ecological environment monitoring center from 1 January 2014 to 31 December 2019. There are nine state controlling air sampling sites to monitor the concentrations of air pollutants in Qingdao. The average daily concentrations of PMhttp://data.cma.cn/), including daily cumulative precipitation (mm), average temperature (℃), average wind velocity (m/s), average atmospheric pressure (hPa), sunshine duration (h), and average relative humidity (%).Meteorological information was downloaded from Meteorological Data Sharing Service System of China  and distributed lag non-linear model (DLNM) in our study24.In ecological research, the delayed effects were often shown in the effects of air pollutants on health outcomes, and the exposure–response curves are often nonlinear26. To control the impacts of meteorological factors, ns functions with 3 df empirically were also adopted in our analysis27. Other covariates such as day of week (DOW) and public holiday in China were included in models.Firstly, a basis model without air pollutants was built. We chose natural cubic spline (ns) to restrict the influence of seasonality and long-term trend, and we used a degree of freedom (df) of 7/yearSecondly, we established cross-basis functions to describe the bi-dimensional exposure-lag-response relationships. For exposure–response relationships, we adopted linear functions. And for lag space, the ns function with three internal knots placed on a logarithmic scale with equidistant values was used. The number of the internal knots was selected according to the minimum Generalized Cross Validation (GCV) score. Given the incubation period of varicella, we selected 20 days as maximum lag period in our research.t is the actual number of adult varicella cases on day t; μt represents the estimated daily adult varicella cases on day t; cb represents the cross-basis function fitting the complex exposure-lag relationships for PM10, PM2.5, NO2, SO2, and O3-8 h, respectively; “lag,3” in cb indicates 3 internal knots placed on a logarithmic scale with equidistant values for lag space; df means degree of freedom; X denotes one of the air pollutants including PM10, PM2.5, NO2, SO2, and O3-8 h.The single-pollutant model was:10, PM2.5, NO2, SO2, and O3-8 h were 70, 35, 40, 60, and 160 μg/m3, respectively28. Relative risks (RRs) were used to assess the lag-response relationships of single-day effects and cumulative effects per a 10-unit increase concentrations of the five pollutants. Besides, to explore the influence of extreme concentrations of air pollutants, we calculated the RRs and 95% confidence intervals (CIs) of varicella at the 2.5th and 97.5th percentiles of concentrations for different air pollutants relative to the class II levels of GB3095-2012 for separate and cumulative lag effect from 0 to 20 days.We used the second levels of Air Quality Standard of China (GB3095-2012) as the references to estimate the influence of air pollution on varicella. There, the reference values for PMp value ≤ 0.05 was regarded statistically significant. All analyses were performed based on R 3.6.3, and involved packages included “dlnm”, “mgcv”, and “splines”.We applied a series of sensitivity analyses to assess the robustness of the results: (1) fitting two or multi-pollutant models for all the air pollutants; (2) changing the df (6–8) per year for time. A two-sided 29, Jinan30, Wuhan and Hongkong31. The peak of adult varicella cases occurred in November and December, and the smaller peak was found in April and May. The season patterns of air pollutants and meteorological factors were also shown in Fig. From 1 January 2014 to 31 December 2019, there were 2, 191 adult varicella cases reported in Qingdao. The female/male ratio was 0.919:1 (3331:3625). In Fig. 10 was 86–68 μg/m3 (from 17.67 to 455.44 μg/m3), 45.7 μg/m3 for PM2.5 (4.38–304.11 μg/m3), 35.89 μg/m3 for NO2 (2.67–116.44 μg/m3), 19.74 μg/m3 for SO2 (2.56–138.78 μg/m3), and 97.57 μg/m3 for O3-8 h (14.88–257.22 μg/m3). The daily means of meteorological factors were 13.92 ℃ for temperature, 1.67 mm for cumulative precipitation, 1008.14 hPa for atmospheric pressure, 5.96 h for sunshine duration, 3.26 m/s for wind velocity, and 68.80% for relative humidity. More details were shown in Supplementary Table From 2014 to 2019, the daily mean content of PM2, SO2, PM10, and PM2.5 were significantly related both to each other. O3 was significantly associated with NO2 and SO2. Besides, the five air pollutants were related to temperature, precipitation, relative humidity, and atmospheric pressure (p < 0.001). Furthermore, to control multicollinearity, we avoided including the pairs whose r ≥ 0.7 in our model simultaneously .Table All the following results were shown in Fig. 3 increase of PM10, there were no significant associations in the lag-specific and cumulative effect curves. In lag-specific effect model, the RR was from 1.000  to the maximum value , and then it gone down to 0.998 . In the cumulative effect curve, RR reached a maximum value with 1.011 .For a 10 μg/m3 concentration of PM2.5 was significantly related to a higher risk of varicella from lag 4 day to lag 6 day . While in the cumulative effect curve, the association was non-significant. The RR rose to the highest value .Per increased 10 μg/m2 per 10 μg/m3 increase and the decreased risk of varicella from lag 15 day to lag 19 day . While in the cumulative effect curve, the association was non-significant. The RR first rose to the maximum value , and then decreased gradually to 0.950 .Significant associations were also found in the relationship between the NO3 increased concentration of SO2 was not significant related to varicella among adults not only in lag-specific effect curve but also in the cumulative effect model. At lag 6 day, in lag-specific effect model, the RR reached the peak value . And in cumulative effect curve, RR gradually decreased to 0.864 .Per 10 μg/m3 increase of O3 concentration, there were no significant associations between O3 and varicella incidence both in lag-specific effect model and in cumulative effect curve. In cumulative effect curve, RR gradually decreased from 1.002  to 0.997 . In cumulative effect curve, RR increased to the peak value , and then decreased.For per 10 μg/m3, the 97.5th percentile of PM2.5 concentration was significantly related to the increased risk of varicella on lag 6 day and lag 7 day . For NO2, compared with 40 μg/m3, the significant associations were found between both at 97.5th and 2.5th percentiles of concentration and varicella on lag 15 day to lag 20 day. The more details were shown in Table 10, PM2.5, NO2, SO2, O3 on varicella  showed a protective effect on the incidence of varicella. As we all know, NO2 was widely used as disinfectant in treatment of industrial water. This reminds us that NO2 may inhibit the spread of the virus attribute to its oxidability.Exposure to high concentration of NO10 and varicella. However, there was only one research assessing the relationship between coarse particulate matter and varicella conducted by Yu et al., which found that high PM10 concentration (> 300 μg/m3) could result in a higher risk of varicella19. The possible causes for this heterogeneity were as follows. Firstly, Yu’s study was conducted in Jiading District, Shanghai but our research was in Qingdao, Shandong province. The difference of geographical areas results in the divergence of meteorological variables, such as temperature, humidity, wind speed and etc. Secondly, the levels of PM10 concentrations were different in these two cities. The maximum concentration of PM10 in Shanghai was higher than that in Qingdao . And in Qingdao, there was only 12 days during 2014–2019 when the concentration of PM10 exceeded 300 μg/m3. Finally, Yu’s research carried out in the whole population, while we only focused on the effects among adults.In our study, no significant association was found between PM10, PM2.5, NO2, SO2, and O3) and the incidence of varicella. Besides, given severe complications and even death can occur among adults, our research focused on adults was necessary and important. Secondly, we explored the influence of exposure to extremely high and low concentrations of air pollutants. Thirdly, our study provided evidence for public health officials to establish a mature environmental monitoring system to predict epidemics of varicella among adults.Some strengths were in our study. Firstly, this is the first study assessing the associations between several air pollutants (PM10, PM2.5, NO2, and SO2, not including other air pollutants. Besides, there is no good way to explore the mixtures effects of air pollutants. Finally, other host and environmental factors which have the significant impacts on the epidemic of varicella were not included in our study, such as population density, host susceptibility, and human serum antibody data.However, some limitations were also found in our study. Firstly, our findings were subject to ecological fallacies. In ecological study, all individuals were supposed to be exposed to the same contents of air pollutants. And causal relationship is difficult to determine, there is often an accompanying relationship between the two variables. Secondly, this study was limited to Qingdao, which may affect the extrapolation of the results. More studies especially multi-city studies are needed to confirm the results. Thirdly, the effect of air pollution is a total effects of various air pollutants. However, we only analyzed PM2.5 was associated with the increased risk of varicella. And the significant relationship was found between exposure to NO2 and the incidence of varicella. This might provide evidence for health authorities to establish a mature environmental monitoring system for varicella among adults.Exposure to PMSupplementary Information.


Perrottetiataronensis from the Dulong Jiang valley in northwestern Yunnan Province, China and the Babulongtan mountain range in northern Kachin State, Myanmar is here described as a new species of the Dipentodontaceae. It is the third species of the genus to be recognized for China and the first to be reported for Myanmar. It is similar to P.alpestriss.s. but differs by characters of its leaf margins, inflorescences, and fruit. The three subspecies of P.alpestris recognized by Hou in “Flora Malesiana” are here recognized as three distinct species, i.e., P.alpestris, P.moluccana, and P.philippinensis on the basis of differences in diagnostic characters and distribution. The report in the “Flora of China” of the Taiwan species P.arisanensis from Yunnan is determined to be incorrect due to misidentification of two specimens at KUN. Perrottetia Kunth is a genus of about twenty species occurring mainly in tropical America and tropical Asia. In addition, two species are endemic to the Hawaiian Islands  Loes., is widely distributed in southern and southwestern China  Loesn. s.s. but differs by several characters given below in the discussion and the key to the Asian and Australasian species of Perrottetia. It is also disjunct from P.alpestriss.s. by over 2500 km. Islands . The genontaceae , now treerteales . Dipentodon Dunn . In the reported . One of rn China , occurriPerrottetia reported that P.arisanensis not only occurs in Taiwan but also in central Yunnan in Eshan Xian 峨山县 and southeastern Yunnan in Xichou Xian 西畴县 (Yuxi Team 玉溪队 89-256 collected 7 July 1989) has been redetermined to be a Rhamnus L., and the specimen from Xichou Xian (Wu Quanan 武全安 7981 collected 7 May 1959) has been redetermined to be P.racemosa. With this correction, P.arisanensis is endemic to Taiwan.The “Flora of China” treatment of Xian 西畴县 . This YuTaxon classificationPlantaeStylommatophoraStreptaxidaeB.M.Barthol. & K.Armstr.sp. nov.6152D7AA-6A69-5057-9C0C-EE4FF0B7312Eurn:lsid:ipni.org:names:77220797-127.6747°N, 98.3015°E, 18 August 2006, Gaoligong Shan Biodiversity Survey 32394 .China. Yunnan Province: Gongshan Xian 贡山县, Dulongjiang Xiang 独龙江乡, Maku Cun 马库村[Taron River], NW facing 30–60° slope, vicinity of Nangza (Pinyin: Laza) 腊咱, W side of the Dulong Jiang valley, ca. 1.3 direct km S of Maku and ca. 3.8 direct km NE of the Myanmar border, 1970 m, Perrottetiataronensis is similar morphologically to P.alpestriss.s. (see Discussion) from which it differs by having much more compact and shorter inflorescences which are sparsely golden tan-tomentose rather than sparsely reddish brown-tomentose, a shorter stipe, leaf margins that are sharply serrate rather than bluntly serrate, and larger fruit when mature.Perrottetiataronensis has been seen. It has two remnant undeveloped flower buds with all other flowers already fallen. In Perrottetia the filaments elongate after the male flowers open, so the filament characters in the observed unopened flower buds of P.taronensis are likely not typical of what they would be during anthesis.) Anthers globular, ca. 0.3 × 0.4 mm. Ovary superior, turbinate, ca. 1.2 × 1.0 mm. Fruit a berry, young fruit green but starting to turn red by July and turning purple and becoming fleshy when mature by August, ca. 5 mm in diam. when mature, usually 4-seeded although occasionally with only 2 or 3 seeds developing, apex emarginate; style ca. 0.2 mm, often deciduous, apically 2-parted. Seeds brown, 1.0–1.5 mm in diam., surface with numerous shallow vertical rugose ridges when dry.Shrubs or small trees 1.5–6.0 m tall, often sprawling, likely dioecious, deciduous. Stems reddish brown, pale brown-tomentose when young, glabrescent. Stipules reddish brown-tomentose, triangular, ca. 1.5 × 0.5 mm, often cauducous, apex long-acuminate. Petioles 0.5–1.0 cm. Leaves alternate, mostly glabrescent; leaf buds and young leaves dactylose Figs , 3, reddtaronensis” refers to the Taron River valley in Myanmar. In China this river is named the Dulong Jiang (Dulong River)  Fig. . The TarPerrottetiataronensis that have so far been made occur in the Ayeyarwady River drainage in both Yunnan Province, China and Kachin State, Myanmar at an elevation range of 1350–1970 m , and among shrubs near river banks. In Myanmar, the habitat of the single collection of a male plant is classified within the Kachin Hills subtropical rainforest ecosystem, a closed-canopy humid lower montane forest type occurring between 700–1500 m , B1ab+2ab, according to the 2 and AOO 319.308 km2 calculated by using GEOCAT , 19 November 1990, Dulong Jiang Investigation Team 独龙江考察队 579 ; Bapo Cun, W side of Gaoligong Shan, W of Gongshan, on the trail from Qiqi 其期 to Bapo in the Dulong Jiang valley, 1900 m, 27.7290°N, 98.3670°E, 17 July 2000, Li Heng 李恒 12931 with Bruce Bartholomew, Philip Thomas, Peter Fritsch, Dao Zhiling 刀志灵, Wang Zhong-lang 王仲朗 & Li Rong 李嵘 ; Bapo Cun, W side of the Gaoligong Shan, along Gamalai He  嘎莫赖河 and Dulong Valley on the trail from Xishaofang 西哨房 to Bapo, 1350 m, 27.7592°N, 98.3377°E, 18 July 2002, Li Heng 李恒 15072 with Li Rong 李嵘 & Dao Zhiling 刀志灵 ; Kongdang Cun 孔当村, W side of Gaoligong Shan, along Dulongjiang Valley on the trail from Bapo to Dizhengdang 迪政当, 1550 m, 27.8773°N, 98.3355°E, 21 Jul 2002, Li Heng 李恒 15156 with Li Rong 李嵘 & Dao Zhiling 刀志灵 ; Xianjiudang Cun 献九当村, W side of Gaoligong Shan along Dulong Jiang Valley on the trail from Kongdang to Dizhengdang, 1560 m, 27.9061°N, 98.3363°E, 23 July 2002, Li Heng 李恒15230 with Li Rong 李嵘 & Dao Zhiling 刀志灵 . Myanmar. Kachin State, Putao District, Naungmung Township, buffer zone of Hkakaborazi National Park, along trail between Maza and Namti, 1763 m, 27.4650°N, 97.6967°E, 18 June 2017, Kate Armstrong 2983 with Thet Yu Nwe, Moe Myint Thu, San Naing Dee, Zaw Naing Tun, Hla Naing Htay & Pa Rang Gang Ken Sar .China. Yunnan Province: Gongshan Xian 贡山县, Dulongjiang Xiang 独龙江乡, Bapo Cun 巴坡村, in the vicinity of Bapo, on the E side of the Dulong Jiang, 1350 m, Perrottetia is now known in Asia and Australasia as having six species of which three occur in China and one in Myanmar. The genus is herein recorded for the first time from Myanmar as it was not mentioned in either P.taronensis is dioecious. This condition is common in the genus, although additional field studies in the spring during flowering are needed for confirmation.As treated here Perrottetiataronensis is most similar morphologically to P.alpestriss.s. (see below) which occurs in Peninsular Malaysia as well as in Indonesia on Sumatra and Java plus nearby small islands. It differs from P.alpestriss.s. by several characters enumerated in the diagnosis above and in the key to the Asian and Australasian species of Perrottetia below. Moreover, P.taronensis and P.alpestris exhibit a north-south disjunction of about 2500 km, with Perrottetia not recorded from intervening continental Southeast Asia  Loes. and P.philippinensis  Loes.) occur in Southeast Asia and Australasia. Although treated as species by P.alpestrissubsp.moluccana (Blume) Ding Hou and P.alpestrissubsp.philippinensis  Ding Hou by Hou in the treatment in “Flora Malesiana” (1962). We follow Loesner in treating all three Southeast Asian and Australasian Perrottetia as separate species. Not only do they differ by several diagnostic characters as can be seen in the key to the Asian and Australasian species of Perrottetia below, but also P.alpestriss.s., P.moluccana, and P.philippinensis are disjunct, as was pointed out by Perrottetiamoluccana occurs on the island of New Guinea and the Cape York Peninsula of Queensland Australia, extending into some of the adjacent islands in the southwest Pacific. Perrottetiaphilippinensis occurs in Indonesia on the islands of Borneo and Sulawesi as well as in the Philippines and on some small adjacent islands. On the basis of diagnostic morphological characters, P.alpestriss.s., P.moluccana, P.philippinensis, and P.taronensis are more similar to each other than this group of four species is to either of the other two Asian species, P.arisanensis and P.racemosa, and these similarities and differences are expressed in the key below. It is hoped that future molecular studies will confirm our interpretation based on diagnostic morphological characters.In addition to Perrottetiataronensis all of which were previously misidentified as Celastrus L. (Celastraceae), Gaultheria L. (Ericaceae), Ilex (Aquifoliaceae), Maesa Forssk. (Primulaceae), or Rhamnus (Rhamnaceae), so it is possible that other specimens are already in herbaria and may come to light, although a search in likely families and genera at KUN as well in the online Chinese Virtual Herbarium (https://www.cvh.ac.cn/) has so far not found additional specimens of this species.We only know of seven collections of


One hundred soil samples were collected from different geographical areas in Khartoum State such as north Omdurman, Toti Island, and Soba. Samples were analyzed for starch hydrolyzing bacteria. Among several bacteria isolated, Bacillus cereus and Bacillus licheniformis were identified as active α-amylase producers. Both bacteria showed a large zone of clearance of 20 mm when grown on starch-agar plates. The identity was conducted using biochemical characterization and confirmed by sequencing their 16S-rDNA. The constitutive nature of amylase was proved by amplification of the amylase gene from the genome of B. licheniformis. The α-amylase activity from the spent medium of B. cereus and B. licheniformis was optimized at pH 8.0 and temperature of 45°C and 65°C, respectively. The α-amylase produced by both bacteria is alkalophilic and thermophilic. The experiments confirmed that B. licheniformis can be a good source of amylase for industrial applications in Sudan.The aim of this study was to isolate some soil bacteria strain that produced  Starch degrading bacteria are mostly important in food, textile, fermentation, and paper industries. The isolation and manipulation of pure culture of starch degrading microorganism from soil have great importance on the biotechnology field. Thus, isolating and manipulating pure culture from various waste materials has manifold importance for various biotechnology industries. The amylase-producing bacteria such as Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. cereus, and B. megaterium and fungi such as Aspergillus Niger, Penicillium, Rhizopus, Cephalosporium, and Neurospora are major amylase-producing microorganisms [α-amylase in the industrial process include the decreased risk of contamination, the increased diffusion rate, and belonging to the genus Bacillus for industrial applications such as B. amyloliquefaciens, B. stearothermophilus, B. cereus, and B. licheniformis [Amylases are starch degrading enzymes that cleave the ase EC 3..11 is enrganisms . Microbirganisms . The therganisms . The advniformis .α-amylase from locally isolated Bacillus species from the soil in Sudan.The aim of this study was to isolate, extract, and purify molecular characterization of thermostable One hundred soil samples were collected from potato fields of Khartoum State: North of Omdurman, Toti Island, and Suba/Sudan. Soil samples were collected from the subsurface of 3 to 4 cm depth with the help of a sterile spatula, then transferred in sterile plastic bags, and brought in aseptic conditions to the laboratory.−1 to 10−4. A volume of 0.1 ml of each dilution was transferred aseptically to a nutrient agar plate. The samples were spread uniformly using a glass rod. Then, plates were incubated at 37°C for 24 hr. The bacterial isolates were further subcultured on the respective media in order to obtain a pure culture. Pure isolates were maintained at 4°C for further studies.Isolation of soil bacteria was performed by serial dilution method . One graHydrolysis of starch was carried out of 10 gm soluble starch in 100 ml distilled water heated in a water bath until dissolved. 20 ml of this solution was mixed in 100 ml of melted nutrient agar poured into the Petri dish after sterilization. A loop full of fresh bacterial culture was picked up by a sterile needle and stabbed onto the agar plate; after 24 hr of incubation at 37°C, the plate was flooded with dilute iodine solution [2PO4 0.05, MnCl2.4H2O 0.015, MgSO4.7H2O 0.25, CaCl2.2H2O 0.05, and FeSO4.7H2O 0.01. The flask was placed on a rotary shaker incubator  at a speed of 7000 rpm at 0°C for 15 min. The supernatant was collected and used for amylase estimation.Freshly prepared inocula were used to inoculate the production medium. For the preparation of inocula, a loop full of bacterial isolate was transferred in 50 ml of inocula medium containing (g/L) starch 10, peptone 10, yeast extract 20, KHThe isolates were identified by colonies morphology with respect to color, shape, size, nature of colony, and pigmentations.Bacterial isolates were stained using gram stain and observed under oil immersion at high power (100X) magnification under a light microscope. Endospore staining, capsule staining, and a motility test were performed to observe the general morphology and motility of the cells .Bacterial isolates were characterized biochemically according to Bergey's Manual  using Voμl of cell lyses buffer  was added and mixed by inverting the vial for 5 min and incubated for 10 min with gentle mixing till the suspension looked almost transparent. 600 μl of isopropanol was layered on top of this solution. The two layers were mixed gently till white strands of DNA were seen and until the solution is homogenous. The strands of DNA were spooled with the help of a pipette tip and transferred into a new vial. 500 μl of 70% ethanol was added to the spooled DNA. The spooled DNA was spun to precipitate DNA at 10,000 rpm for 10 min, the supernatant was discarded. The pellet was air-dried . 50 μl of 1X TE was added and the pellet was suspended (incubated for 5 min at 55–60°C to increase the solubility of genomic DNA).Colonies from a single streak on the agar plate were scraped and suspended in PBS and centrifuged. The pellet obtained was dispersed. 600 µl at 94°C for 5 min followed by 35 cycles  and a final extension at 72°C for 5 min. Finally, the PCR products were subjected to electrophoresis in 1% agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light. The length of the amplicon was approximately 1.5 kb.Amplification of the 16S rRNA gene was performed using the primers sets of 16S rRNA fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (5′-ACGGCTACCTTGTTACGACTT- 3′) . PCR wasα-amylase gene-specific primers; F: 5′-ATTGGTAACTGTATCTCAGCTTGAAGA-3′ and R: 5′-TCCGTCCTCTCTGCTCTTCTATCTT-3′. PCR was conducted in a total volume of 50 µl at 94°C for 5 min followed by 35 cycles  and a final extension at 72°C for 7 min. Finally, the PCR products were subjected to electrophoresis in 2% agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light. The length of the amplicon was approximately 1.7 Kb (The isolated DNA samples were amplified using y 1.7 Kb .http://www.ncbi.nlm.nih.gov/). Neighbor-joining phylogenetic trees were constructed based on 16S rRNA sequences using ClustalW.The amplified DNA was visualized by gel electrophoresis and the PCR products were purified using the solution for Gel Extraction kit (Omega Bio-tek); the purified products were subjected to sequencing by the Sanger technique . The most similar bacterial species were found in the GenBank by using BLAST search  method was used to determine nd Stein . UN inocProtein was measured by the method of Lowry et al.  with bovPurification of amylase enzyme was achieved by ammonium sulphate precipitation followed by dialysis. 100 ml of cell-free extract was saturated with centrifuged at 7000 rpm for 15 min. The supernatant was collected and saturated up to 0–30 and 30–80% with ammonium sulphate. Then, the content was centrifuged at 7000 rpm for 15 min and the pellet was collected for further analysis. The enzyme mixture was transferred in a dialysis bag with space size 70 cm and immersed in phosphate buffer pH 7 at 4°C for 24 hr. The buffer was continuously stirred using a magnetic stirrer throughout the process. The buffer was changed three times during the process in order to obtain proper semipurification.Thermal inactivation of the dextrinogenic activity of the enzyme produced from the bacteria strains was performed by the enzyme incubation at different temperatures for 15 min at ranges of 35, 45, 55, 65, and 75°C.B. licheniformis and B. cereus was conducted at temperatures of 4, −20, and −80°C for 24 hr.Storage inactivation of the dextrinogenic activity of the enzyme produced from α-Amylase activity enzyme was tested by different pH ranges of 3.5 to 5.5 using acetate buffer, pH of 6.0 to 7.5 using phosphate buffer, and Tris-HCl for pH 8.0 to 8.5.Bacillus sp. Screening of amylolytic bacteria was performed on starch media treated with iodine solution and the colony which showed the disappearance of the blue color around it was selected. In the present study, 66% of the samples showed wide zones around colonies of 3.0, 2.5, 1.5, and 1.25 mm.Thirty samples out of 100 (30%) isolates were found to be B. licheniformis strain and negative for B. cereus strain.Microscopical examination showed that 66% of isolates were gram positive and positive for motility test, citrate test, growth on normal saline of 6.5%, starch hydrolysis, and VP test. However, the growth at 55°C showed positive for Bacillus strains [B. licheniformis while the temperature of 45°C was optimal for B. cereus (The temperature ranges of 35–45°C were reported to be nominated for the biosynthesis of amylases by  strains . The cur. cereus .Bacillus strain isolated from soil [α-amylase by B. licheniformis (15.959 U/mL/min) and B. cereus (247.20 U/mL/min) was found to be optimum when the initial pH of the fermentation medium was maintained at 8.0. Further increase or decrease in pH resulted in a gradual reduction of amylase production  and B. cereus (accession number LC506178), respectively, based on the first five identities  of similarity with the known sequences in NCBI databases was compared using Basic Local Alignment Search Tool (BLAST). Maximum Likelihood (ML) phylogenetic tree for determination of the relationship between our isolates and other reference strains was drawn using MEGA 7.0 software. Phylogenetic analysis indicates that the partial sequence of 16S ribosomal RNA gene of our isolates (L1 and S2) showed that the α-amylase enzyme was achieved by ammonium sulphate precipitation followed by the dialysis technique. The activity of the crude α-amylase from Bacillus licheniformis and B. cereus arrived to 247.20 and 15.959 U/ml, respectively, but decreases at each purification step till reaching 8.47 and 0.03 U/ml indicating that the purification step was corrected and enabled to increase specific activity to 5.26 and 0.02 U/mg with purification fold reaching to 0.32 and 0.04 and enzyme recovery arrived at 3.4 and 0.002%, respectively; this decline in activity is imputed to freezing, thawing, and freeze-drying processes  in 15 min indicated that the incubation temperature of 45°C is optimum for B. licheniformis and B. cereus growth. The enzyme was stable for 15 min at a temperature of 45°C while B. cereus lost 100% of its activity at 75°C for 15 min but the B. licheniformis activity was reduced to 70% at 75°C for 15 min (The results of thermal stability of the dextrinogenic activity for two r 15 min .B. licheniformis and B. cereus were found to be with no activity on −80°C for 24 hr. The enzyme was stable for 7 days at a temperature of 4°C for B. licheniformis without loss of enzyme activity while for B. cereus, the enzyme was stable up to 5 days with only 10% loss in activity (Results of storage inactivation of the dextrinogenic activity for activity .B. licheniformis at −20°C was stored for 2 days with 30% loss in activity but B. cereus was stored for 3 hr with 70% loss in amylase activity. At −80°C, both B. licheniformis and B. cereus amylase activity were lost 100%.B. cereus and B. licheniformis (Results indicated that pH of 8.0 was the best for the highest enzyme activity for both niformis .α-amylase produced by B. licheniformis and B. cereus, respectively. This is in line with Mishra et al. [B. amyloliquefaciens, B. subtilis, B. licheniformis, and B. stearothermophilus are among the most commonly used Bacillus sp. that produce amylase at temperatures 37–60°C. Our study indicates that optimum α-amylase production occurred in pH 8.0 for both Bacillus sp. This finding is consistent with the finding of Alkando and Ibrahim [B. licheniformis gave maximum α-amylase activity of 0.7947 U/mg/ml/min at a pH of 8.0. Furthermore, the present result agrees with those studies reported for B. licheniformis [B. subtilis JS-2004 [B. Subtilis BI19 [The present study revealed that 65°C and 45°C were optimum temperatures for a et al.  who repo Ibrahim  who reponiformis , B. subt JS-2004 , and B. lis BI19 .α-amylase for B. licheniformis and B. cereus was observed at 24 h after inoculation. This may be attributed to the organism which entered the stationary phase and fermentation approached its end point [In the current study, the highest production of nd point . Maximumnd point . Similarnd point  and Biplnd point . The curα-amylase produced by B. licheniformis was more stable at a high temperature compared to B. cereus. A similar finding was observed by Alkando and Ibrahim [B. licheniformis lost 72% of its activity at 90°C for10 min. Moreover, for B. licheniformis, the amylase enzyme was stable for a week at 4°C without loss of activity unlike B. cereus which lost 10% of its activity in the same condition. A similar result was reported by Kumari et al. [The present study indicates that the activity of  Ibrahim  who repoi et al. .Bacillus licheniformis and Bacillus cereus secreted thermophilic and alkaliphilic α-amylase under optimized culture conditions. In addition, it was observed that B. licheniformis was a better producer than B. cereus. The experiments confirmed that B. licheniformis can be a good resource of thermophilic and alkaliphilic α-amylase for industrial applications in Sudan.It could be concluded that


Phase shifter isone of the key elements of quantum electronics.In order to facilitate operation and avoid decoherence, it has tobe reconfigurable, persistent, and nondissipative. In this work, wedemonstrate prototypes of such devices in which a Josephson phaseshift is generated by coreless superconducting vortices. The smallnessof the vortex allows a broad-range tunability by nanoscale manipulationof vortices in a micron-size array of vortex traps. We show that aphase shift in a device containing just a few vortex traps can bereconfigured between a large number of quantized states in a broad range. The autonomous  operation is needed to avoid decoherence in qubits,6 as well as for realization of complementary digital electronics16 and nonvolatile memory.22 The tunability is requiredboth for qubit12 and for memory operation. Simultaneously, phase shifters shouldbe small, to facilitate downscaling. The combination of persistency,tunability, and compactness is difficult to realize. Depending onthe number of achievable states the phase shifter can be switchablebetween two (0/1) states, reconfigurable between several states, orcontinuously tunable.Electronic devicesoperate with certain electronic degrees of freedom:conventional electronics with the charge, spintronics with the spin,and quantum electronics with the phase of the electron wave functions.A phase shifter is an important element of quantum devices such asqubits.x) ≠ 0 (0 – φjunctions). The π JPS is most commonly needed, for example,for bringing a qubit to the degeneracy point,4 for complementary Josephson electronics,16 and for maximum distinction between 0/1 states in memory cells.20 Several ways of creation of JPS are known. Aspatial phase variation within JJs can be introduced by inhomogeneitiesand uneven current distribution.29 π-junctions with a fixed φ = πphase can be realized using hybrid superconductor/ferromagnet (S/F)structures32 and unconventional superconductors with a sign-reversalorder parameter.35 φ-junctionswith an arbitrary JPS can be realized using inhomogeneous SFS JJs,37 or JJs with a strong spin–orbit coupling.24Superconducting electronics typicallyoperates with the Josephsonphase difference φ. Usually φ = 0 in the absence of appliedcurrent or magnetic field. However, unconventional Josephson junctions(JJs) may provide either a fixed Josephson phase shift (JPS) φ≠ 0 (φ-junctions) or an in-built spatial phase variationalong the JJ φ(26). The tunability can be achieved in Josephsonspin-valve-type devices with several F-layers40 or in SFS JJswith a strong spin–orbit coupling.24 However, it is not clear if SFS JJs are suitable for qubits, becausemagnetism is considered as one of the main sources of decoherence.42 Therefore, flux qubits utilize an additional “trap loop”with trapped flux quanta, Φ0, nearby the qubit, whichoffsets the qubit state.6 The offset flux, Φ, is switchableby magnetic field and adjustable by the loop/qubit geometry. Similarphase shifters have been used for digital RSFQ electronics.15 Recently it was shown that an Abrikosov vortex can induce a JPSin nearby JJs in the full  range.44 Since vortices can be easilymanipulated  by magnetic field, H,47 current, I,49 and light,51 they can be used for creationof memory cells20 and tunable phase shifters.The majority of phase-shifted JJs are either persistentbut nottunable  ortunable but not persistent  – φv(0) = Θv. The redline in v(x) for an antivortexplaced in the middle of the electrode, xv = 0.5L, at a distance zv = 0.1L to the JJ. In v.44 It is seen that a single vortex can produceany phase shift in the full range . In what follows,we utilize this phenomenon for creation of a reconfigurable Josephsonphase shifter.Here 52 Details of sample fabrication and experimentalprocedurescan be found in the Supporting Information. First, we demonstrate operation of the simplest device containingone planar JJ with L ≃ 4.6 μm and onevortex trap (a hole with a diameter 30–50 nm) at xv ≃ L/2 and zv = 500 nm ≃ 0.1L. T ≃ 4.4 K in the absenceof the vortex (e) and with a trapped antivortex (f). The antivortexis clearly visible as a dark spot at the trap. Vortex-induced JPSleads to a distortion of the critical current modulation pattern, Ic(H).44Ic(Φ) patterns without a vortex (blue)and for the JPS from panel b, created by an antivortex at zv = 0.1L (see the Supporting Information). The Ic(H) distortion uniquely depends on theJPS and can be used for estimation of φv.44Studied devices contain Nb-based planar JJs.Ic(H) modulationfor this JJ measured in the vortex-free state, Ic(H) pattern after trappingan antivortex, Ic(H), in good agreement with the simulationin Ic(H) patterns, as described in refs . 44 Vortices are manipulated (introducedand removed) by short current pulses, Ip.20 The top panel in H = 0. The bottom panel showsthe corresponding time-dependence of the junction resistance. It isseen that we can controllably and reproducibly switch between vortex, V = 1, antivortex, V = −1, and vortex-free, V = 0, states. The same procedure can be done in an appliedfield H ≠ 0, which helps to achieve more complexstates. We will identify them as , where Vi is the vorticity in the trap i, counted from topto bottom in V = |V1| + |V2| + |V3| + |V4|. Black symbols in Ic(H) patternsof both JJs for different single vortex, |Vi| ≤ 1, configurations. Red lines demonstratenumerical fits, from which we deduce Δφv.Ic(H) in J1 is practically unaffected, Δφv ≃0, but in J2 there is a significant JPS, Δφv ≃ −1.29π. This is a manifestation of the state with a vortex in the bottom trap, at a maximum distance fromJ1. v ≃ −0.78πin J1 and −1.65π in J2. It corresponds to the state with vortices in the two lower traps. v ≃ −2.15πand −2.67π, and the  state with Δφv ≃−2.36π and −2.67π. Moredetails about vortex state identification can be found in ref  modulations of the SQUID without (black)and with (red) an antivortex in the trap. It is seen that the Ic(H) modulation patterns areshifted by approximately half a period, indicating that the phaseshift is close to π.A vortex can induce a phase shift not only in aJJ but also ina device, such as SQUID. v, which depends on the device geometry. As seenfrom xv/Lx = 0.5 and zvi/Lx = 0.05, 0.16, 0.27, 0.4. The correspondingpolar angles are Θvi = 0.937π, 0.803π,0.685π, and 0.570π. We include multiple quantized vorticesin the analysis so that each trap could be either empty or containvortices and antivortices with |Vi| ≤ V. For V = 1 there are eight states (fourvortex and four antivortex) with phase shifts given by ± Θvi. The combinatoric number of states increases rapidly withthe total vorticity and reaches 152 for V = 4, whilethe range of the phase shift increases linearly with V. Green, blue, and red lines in V ≤ 2, 3, and 4, respectively. In particular, redlines represent 276 states with V ≤ 4, whichis the sum of the states with V = 1, 2, 3, and 4.It is seen that the states are very densely distributed in the range. Therefore, reconfigurability of sucha phase shifter is similar to continuous tunability. However, allof these states are distinct, quantized, and persistent.Thus, we have demonstrated thatvortices can induce JPS in nearbyjunctions and devices. As shown in zv < 0.4Lx. For Lx = 2 μm,this is only 800 nm. The compactness is facilitated by the small vortexsize, ∼100 nm, which enables significant JPS variation by ananoscale vortex manipulation. This is the main advantage with respectto macroscopic trap loops, employed for offsetting qubits so far.6 Such loops allow only on–off switching of a certain phaseshift, while vortex-based phase-shifters, presented here, can be almostcontinuously tuned in a broad range without increasing device sizes.A remarkablefeature of vortex-based phase-shifters is a combinationof the broad JPS range with compactness. From 45 immobilizing vortices, and removes vortex coresand associated quasiparticles. Therefore, such vortices should notaffect coherence times in quantum devices.Finally we want to note that Abrikosov vortices are unwanted inquantum devices because they can cause decoherence as a result ofextra dissipation in the normal core or due to vortex motion. Here,we employ coreless vortices trapped in holes several times largerthan the coherence length of Nb. This leads to a very effective pinning,Lx), position of vortextraps  and vorticity (Vi). The main advantages of such devices are (i) compactness and (ii)broad range of tunability, facilitated by the small vortex size (vorticesrepresent the most compact magnetic object in a superconductor); (iii)persistent and nonvolatile operation caused by the quantized natureof vortices; (iv) low dissipation and decoherence due to usage offirmly trapped coreless vortices; and (v) easiness of vortex manipulation,which facilitates various ways of operation. We argue that this providesa unique opportunity for creation of compact, tunable, and persistentphase-shifters and phase batteries both for digital and quantum electronics.To conclude, we haveshown that vortices can be used for creationof reconfigurable Josephson phase shifters. We demonstrated prototypesof such devices in which the phase shift is controlled by geometry(


Echinococcus granulosussensu lato (s.l.) causing human cystic echinococcosis (CE) in Europe during the period 2000–2021. As these species and genotypes are characterized by genetic, animal host and geographical differences, studying the E. granulosuss.l. complex is epidemiologically relevant.This study aimed to fill a gap of knowledge by providing a quantitative measure of molecularly identified species and genotypes belonging to E. granulosuss.l. at the genotype/species level as a causative agent of human CE cases in selected European countries.A systematic review (SR) was conducted on the basis of both scientific and grey literature considering primary studies between 2000 and 2021 in four databases. From a total of 1643 scientific papers, 51 records were included in the SR. The main inclusion criterion for this study was the molecular confirmation of E. granulosussensu stricto (s.s.), 130 (21.7%) as E. canadensis cluster (G6/7 and G10), 7 (1.2%) as E. ortleppi (G5), and 2 as E. vogeli (0.3%). Three geographical hotspots of human CE caused by different species of the E. granulosuss.l. complex were identified: (1) E. granulosuss.s. in Southern and South-eastern Europe ; (2) E. canadensis (G6/7) in Central and Eastern Europe; (3) E. ortleppi in Central and Western Europe. This SR also identified data gaps that prevented a better definition of the geographical distribution of the Echinococcus granulosuss.l. species complex in Europe: western Balkan countries, part of Central Europe, and Baltic countries.Relevant data were obtained from 29 out of 39 eligible European countries. This SR identified 599 human molecularly confirmed echinococcal cysts: 460 (76.8%) identified as E. granulosuss.l. species complex and their potential clinical impact on humans.These results mandate longitudinal, multi-centre, intersectoral and transdisciplinary studies which consider both molecular and clinical epidemiology in animals and humans. Such studies would be valuable for a better understanding of the transmission of the The online version contains supplementary material available at 10.1186/s13071-022-05197-8. Echinococcus spp. infections, CE is the most prevalent at the global level, causing high morbidity and relative mortality among human populations . Finally, a total of 51 records were included in the SR for data extraction  member states], namely: Albania, Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Finland, France, Germany, Greece, Hungary, Italy, Lithuania, Luxembourg, Norway, Poland, Portugal, Republic of North Macedonia, “Republic of Kosovo”, Republic of Moldova, Romania, Serbia, Slovak Republic, Slovenia, Switzerland, Spain, the Netherlands, the UK and Ukraine. Data where searched but not obtained from the following ten eligible countries (eight were EU member states): Belarus, Czech Republic, Denmark, Estonia, Ireland, Latvia, Malta, Montenegro, Republic of Cyprus, and Sweden.Echinococcus species and/or genotypes, and if these specimens were assumed to be locally acquired or not.Additional file E. granulosuss.l. circulating between countries in Europe, 12 (2%) belonged to patients of unknown country of origin  and 78 (13%) belonged to patients of unknown imported country of origin , while 96 (16%) were imported from non-European countries.This SR identified a total of 599 human CE cases that were molecularly identified in European studies  genotype G1, 31 (10.8%) genotype G3 and 141 (48.9%) ungenotyped], 119 (28.8%) as E. canadensis cluster  and 6 (1.5%) as E. ortleppi (G5).Among 413 human CE cases molecularly confirmed in Europe, the nationality of 350 (84.7%) cases coincided with country of diagnosis, whereas for 63 (15.3%) it did not. Considering these 413 human molecularly confirmed CE cases circulating in Europe, 288 (69.7%) were identified as n = 46), Morocco (n = 13), Algeria (n = 6), Iraq (4), Tunisia (n = 3), China (n = 2), Ghana (n = 2), Iran (n = 2), Kazakhstan (n = 2), Lebanon (n = 2), Russia (n = 2), Suriname (n = 2), Afghanistan (n = 1), Armenia (n = 1), Chile (n = 1), Eritrea (n = 1), India (n = 1), Mali (n = 1), Mauritania (n = 1), Nigeria (n = 1), Syria (n = 1) and Thailand (n = 1). Among these 96 CE cases from non-European countries, 88 (91.7%) were identified as E. granulosuss.s., while 6 (6.2%) as E. canadensis   genotype G1, 44 (9.6%) genotype G3 and 251 (54.5%) ungenotyped], 130 (21.8%) as E. canadensis cluster , 7 (1.2%) as E. ortleppi (G5) and 2 as E. vogeli (causing NE) (0.3%)  were identified as E. granulosuss.s.  based on 460 specimens  based on 130 specimens. Genotype G10 was only documented in one locally acquired case from Finland, confirming rareness of this species infection in humans. Luxembourg and Switzerland, considered non-endemic countries for CE, were the only countries reporting only imported cases of genotype G6/7. Considering countries with cases considered only locally acquired or both locally acquired and imported, E. canadensis G6/7 was the dominant Echinococcus species  was determined on the basis of seven specimens, therefore confirming the rareness of this species infection in humans , Poland (n = 1) and Switzerland (n = 1). Germany documented one imported case of unknown country of origin. This figure identifies Central and Western Europe as hotspots for the few human CE infections caused by E. ortleppi, areas where cattle raising is practised  of which were from 16 European countries [E. granulosuss.s. , 184 (11.07%) as E. canadensis G6/7 , 6 (0.36%) as E. ortleppi , 1 (0.06%) as E. canadensis genotype G8 (genotype not identified in our study) and 1 (0.06%) as E. canadensis G10 .This SR provides the first comprehensive view and synthesis of the genotypes and species belonging to the ountries . In thisE. granulosuss.s. as the main driver of CE infections in humans. However, the proportion of cases attributed to E. canadensis (G6/7) in Europe is almost double that detected worldwide (21.8% versus 11.1%) [E. canadensis G6/7 in this study, as well as that at the worldwide scale, could be biased by sampling effort. In fact, the number of molecularly confirmed human samples probably does not reflect the actual numerical burden of CE in specific endemic countries , but rather the presence of experts in those countries [E. canadensis G6/7, but also other species and genotypes, in Europe cannot be ruled out.In line with the review by Alvarez-Rojas and colleagues , the curs 11.1%) . It shouountries , 57. HowE. ortleppi: six considered as locally acquired in France, Poland and Switzerland, and one considered as imported in Germany . To the best of our knowledge, 16 additional CE cases  caused by E. ortleppi have been detected in humans globally from Argentina (n = 5), Brazil (n = 1), China (n = 2), India (n = 1), Mexico (n = 1), the Netherlands (n = 1), South Africa (n = 1) and Vietnam (n = 4) [This study identified seven human cases infected with  (n = 4) –69.E. canadensis genotype G10 from Finland [n = 1), Far East Russia (n = 1) and Mongolia (n = 2) [E. canadensis genotype G8 was identified in this study from Europe. Only one human infection has been globally documented in the past from Alaska [Our study identified only one locally acquired case of  Finland . To the  (n = 2) –73. No cm Alaska .E. granulosuss.l. on humans in Europe may reflect: (1) the presence of high numbers of the main IH , which maintain the parasite species life cycles and consequently the biomass of viable eggs contaminating the environment; (2) the hygiene conditions of rearing animals (e.g. home slaughtering versus controlled slaughtering) and type of husbandry (e.g. intensive versus backyard practices), which may increase odds of transmission of Echinococcus species to humans; (3) deworming practices of dogs DH that release eggs, the parasite infective stage for humans. In this context, the few human infections detected in Europe caused by E. ortleppi and E. canadensis G10 may be explained by the above-mentioned low infection pressure of these Echinococcus species. In fact, the improved hygiene conditions of cattle breeding during past decades in Europe  decreased the presence of E. ortleppi in animal hosts (e.g. the disappearance of this parasite species in Switzerland). On the same note, the sylvatic cycles of genotypes G8 and G10 may explain the low exposure of humans and, consequently, the few documented human infections.The main drivers of infection pressure of species of E. granulosuss.l. species to humans has already been debated by several authors [E. granulosuss.l. is not relevant in daily clinical practice, as it does not influence the clinical management, treatment and follow-up of human CE cases. Nevertheless, it must be pointed out that the identification of genotypes and species of E. granulosuss.l. circulating in humans is important in identifying the reservoir species of the parasite circulating in humans, and consequently the possibility of assessing source attribution and risk factors that should be targeted for implementing surveillance and control programmes. Moreover, different parasite biological potential due to different prepatent periods of worms in the DH and potential difference in efficacy of EG95 vaccine in the IH should be considered [The potentially different infectivity and pathogenicity of the  authors , 75, 76.nsidered .E. granulosuss.s. and E. canadensis genotypes, as reported by the authors of the studies included in this SR. The limit of nationality, the time lag (months/years) between the event of Echinococcus spp. infection and the eventual appearance of symptoms make it almost impossible to trace the source and place of human CE infection. Assuming this level of uncertainty, the nationality of patients was used as a proxy to define the country of infection, i.e., if these Echinococcus species/genotypes were circulating or not in a given European country. As for the study of Alvarez Rojas [E. canadensis (G7), 1 E. ortleppi and only 2 E. granulosuss.s. In Poland, E. granulosuss.s. was identified for the first time in 2017 in only one sheep [E. canadensis G7 has been widely documented in animals and humans [E. granulosuss.s., the first was of Kazak nationality , and the second had a history of travel 2 years before in Turkey (another highly endemic country for E. granulosus s.s). According to our case definition, the first case was considered not originated in Europe but in Kazakhstan, while the second, though the patient was probably exposed in Turkey, was considered as locally acquired in Poland [Finally, it is worth noting two potential limits of this study, namely the assumption about the nationality of the patients and the identification of ez Rojas   can be contacted to coordinate these analyses (Commission Regulation 776/2006).Having illustrated the relevance of collecting molecular data, the standing question is what path clinicians should follow when handling parasitic samples from CE cases. The authors suggest for this task to refer to the NRL for Parasites for E. granulosuss.l. species have largely been generated on the basis of mitochondrial cox1 gene sequencing [E. granulosuss.l. species have been made available in literature, as well as new molecular tools for genotype differentiation (G1 versus G3 and G6 versus G7) within this species complex [Moving towards a standardized genotyping approach to be integrated into the national public health system is the way forward. During the past 30 years, molecular typing data of quencing . Recentlquencing  and a vaquencing  to diffe complex , 81.Echinococcus isolates, keeping together molecular and clinical epidemiology in animals and humans. Strengthening such studies will lead to a better understanding of the transmission of the E. granulosuss.l. species complex and their potential clinical impact to humans.Finally, we encourage longitudinal, collaborative, multi-centre, intersectoral and transdisciplinary research in Europe and beyond to perform molecular analysis on E. granulosuss.l. species and genotypes infecting humans in Europe. The study also identified a series of gap of knowledge at the country level on the presence and distribution of E. granulosuss.l. in humans. In particular, no genetic data were available for several endemic or importing countries  or the data were scanty . Moreover, this SR identified two grey zones to better define the geographical distribution of the E. granulosuss.l. complex in Europe: one overlapping between the distribution of E. granulosuss.s. and E. canadensis G6/7 and the other in the northern European geographical distribution of E. canadensis G6/7, where it is not clear which are the main species of E. granulosuss.l. infecting humans. The first grey zone corresponds to the western Balkan countries  and part of Central Europe (in particular Czech Republic and Slovakia), whereas the second zone was identified in the Baltic countries (in particular Latvia and Estonia), where more sampling effort is needed to understand which parasitic species represents a public health problem, resulting in targeted control measures. An additional advance in this direction would be represented by the systematic implementation of shared molecular tools (using dedicated loci) in the field of epidemiology for the molecular identification of causative species belonging to E. granulosuss.l.This SR provided new data at the diversity and distribution of Additional file 1: Table S1. PRISMA 2020 Checklist.Additional file 2: Table S2. Summary of data extracted from publications reviewed in this study.