There is still a certain gap between the effective implementation and requirements of sepsis bundle. Our aim is to establish the clinical nursing pathway of the cluster treatment of septic shock in the Intensive Care Unit and promote effective implementation of the cluster treatment of septic shock.By means of evidence-based method, quality control index requirements and on-site investigation, the implementation process of clinical nursing pathway of the cluster treatment within 6 h of diagnosis of septic shock was established.P < 0.05), which was significantly improved in the experimental group compared with the control group.After the implementation of clinical nursing pathway, the completion rate of septic shock cluster treatment was 81.4% (66.4%) in 1 h, 89.4% (77.0%) in 3 h, 95.5% (82.3%) in 6 h (The clinical nursing pathway of septic shock cluster treatment is guided by evidence-based nursing, which emphasizes standardization and standardization of septic shock cluster treatment nursing under the guidance of the guideline, and can promote the effective implementation of septic shock cluster treatment, significantly improve efficiency of septic shock treatment and the quality of medical care. Septic shock is also known as infectious shock. The study of ICON provided global epidemiological data on 10,069 Intensive Care Unit (ICU) patients, confirming that 2973 29.5%) patients had sepsis during admission or ICU stay. Among sepsis patients, the ICU mortality rate was 25.8%, and the hospital mortality rate was 35.3%, which was much higher than that of the general ICU population  [% patientClinical pathway is to formulate a standardized workflow according to the expected length of hospital stay of a certain disease, and conduct procedural and standardized management of medical and nursing behaviors, so as to improve the quality of medical care, shorten the length of hospital stay, and reduce medical costs . It turnP > 0.05) between the two groups in terms of age, gender, APACH II score, primary disease, etc., and they were comparable. See Table Two hundred twenty-six patients with septic shock admitted to our hospital from March 2017 to March 2020 were divided into control group and treatment group with the random number table, with 113 cases in each group. The control group was routinely treated with sepsis bundle, while the treatment group was treated by clinical nursing pathway of sepsis bundle. There was no statistically significant difference  ≥ 65mmHg , 10.The completion rate of 3-h sepsis bundle for septic shock: the completion rate of 3-h sepsis bundle for septic shock refers to the completion within 3 h after the diagnosis of septic shock: ➀ measure the concentration of lactic acid; ➁ blood culture before antimicrobial therapy; ➂ apply broad-spectrum antimicrobial drugs; ➃ apply 30 ml/kg crystalloid solution for target resuscitation under hypotension or lactate ≥ 4 mmol/L.2 should be measured immediately under continuous hypotension after septic shock or lactate ≥ 4 mmol/L volume; ➂ lactate level should be measured repeatedly in patients with initial lactic acid higher than normal.The completion rate of 6-h sepsis bundle for septic shock: based on 3-h sepsis bundle for septic shock, the 6-h sepsis bundle for septic shock should add: ➀ vasopressor should be immediately applied when hypotension has poor effect on target resuscitation; ➁ CVP and ScvOThe construction of pathway was under the guidance of evidence-based medicine, international guidelines, The Medical Letter 252 (2015) and the notification on printing and distributing six professional quality control indicators of anesthesia, etc. (2015 version), by National Health Commission of PRC. The clinical nursing pathway of sepsis bundle in septic shock is shown in Table By reviewing the hospitalized medical records of patients with septic shock in our department, we can understand the current status, including the number of hospitalization days, costs, medical care content and implementation.Set up a clinical nursing pathway group in the department, invite the department director, doctors and nursing team leader to discuss together, and formulate the clinical nursing path framework for the sepsis bundle of septic shock.2) ≥ 70% or mixed venous oxygen saturation (SvO2) ≥ 65%, urine volume ≥ 0.5 ml/kg/h after diagnosis of septic shock 6 h, the patient’s peripheral warm, skin mottling turns better.Each time node completes all items as required, and the patient’s mean arterial pressure (MAP) ≥ 65 mmHg, central venous pressure (CVP) 8–12 mmHg, central venous oxygen saturation , and t test was used for comparison between groups. P < 0.05 shows the difference is statistically significant.SPSS 17.0 statistical software was used for data analysis. Count data were tested by p < 0.05, the difference was statistically significant. See Table After the implementation of the clinical nursing path of sepsis bundle for septic shock, the target completion rate of 1-h sepsis bundle of septic shock increased from 66.4 to 81.4%, and the completion rate of 3-h sepsis bundle of septic shock increased from 77 to 89.4%, the completion rate of 6 h sepsis bundle of septic shock increased from 82.3 to 95.5%, the treatment group was significantly higher than the control group, Septic shock is a medical emergency and should be treated and resuscitated immediately. The primary task of doctors and health care providers is to protect patients from harm, and sepsis bundle can help us to do this . HoweverAs an industrial management method, critical path was widely applied in American industry around 1950. In the medical field, DRG/PPS (Diagnosis Related Groups/Prospective Payment Systems) was introduced into the United States in 1983. Then, in 1985, Karen Zander from New England Medical Center in Boston introduced the method of critical path into the nursing of inpatients. In Japan, since about 1992, the clinical pathway has been centered on the Japanese medical management association and the Japanese clinical pathway society, as the enrichment of informed consent, the development of team medicine, the medical reform centered on patients, the saving of medical resources and the improvement of safety have been recognized and carried out to improve the medical quality. Clinical pathway is very necessary to promote efficient diagnosis and treatment. It can provide standard treatment and management systematization, improve the coordination of medical staff, save medical resources, and improve medical safety and patient satisfaction. Compared with the doctor-centered clinical pathway, the clinical pathway with the ward supervisor as the center has more continuity and can become a sound path activity. Clinical nursing pathway makes nurses’ work change from passive to active, cooperating with doctors, the whole process of diagnosis and treatment can reflect the opinions of nurses. Clinical nursing pathway can also deepen nurses’ understanding of the significance and results of examination or disposal, reduce medical errors, facilitate early detection of abnormalities, continue nursing care even for new nurses, promote team medical care, enhance communication with patients, and improve the trust of doctors and patients , 15.2 monitoring can not be implemented due to femoral vein catheterization in emergency or other reasons or patients’ introduction of peripherally inserted central catheter [After the implementation of the clinical nursing pathway of sepsis bundle for septic shock, the completion rate of 1-h, 3-h, 6-h sepsis bundle of septic shock was significantly improved, but it did not reach 100%, The reasons are as follows: patients’ economic difficulties or doctors’ working habits lead to difficulties in target monitored volume recovery; patients have received rehydration treatment before they are transferred to ICU; CVP measurement and ScvOcatheter . To treacatheter .The clinical nursing pathway of sepsis bundle for septic shock is guided by evidence-based nursing. It emphasizes the standardization and standardization of nursing care under the guidance of the guideline, which can promote the effective implementation of sepsis bundle for septic shock and improve the satisfaction of doctors to nursing behavior and nursing work. It has important application value in promoting the unity of doctors and nurses, mobilizing the enthusiasm of nurses, reducing errors and improving the level of hospital service. It can significantly improve the treatment efficiency of septic shock and the quality of medical care.


Bacillus from the sake brewing process at Shiraki Tsunesuke Sake Brewery in Gifu, Japan to determine whether there is strain specificity at individual sake breweries. After distributing the isolates into seven groups, we observed that at least two groups (68 isolates) were kuratsuki bacteria at Shiraki Tsunesuke Sake Brewery. The kuratsuki Bacillus isolates were collected from different samples at the early and late stages of sake brewing in 2021 and 2019, respectively. These results showed that kuratsuki Bacillus entered the sake brewing process at this location. These kuratsuki Bacillus isolates had a high ethanol tolerance. Our previous paper showed the existence of kuratsuki Kocuria at Narimasa Sake Brewery in Toyama, Japan, but this study demonstrated that it is not found at Shiraki Tsunesuke Sake Brewery. Therefore, each sake brewery has specific kuratsuki bacterial strains, which are isolated with high frequency and contribute a specific flavor or taste to each sake brewery.We collected 92 isolates belonging to the genus  Aspergillus oryzae is a fungus that converts rice starch into sugars, and then another fungus (yeast), Saccharomyces cerevisiae, converts the sugars into ethanol during sake brewing  lactic acid bacteria have been used for preventing the growth of other bacteria, which can lead to sake spoilage [kuratsuki lactic acid bacteria grow and produce lactic acid [S. cerevisiae. In contrast, some highly ethanol-tolerant lactic acid bacteria, for example Lactobacillus fructivorans, spoil sake [Lactic acid treatment is performed before the addition of entation . Kuratsuspoilage . In the tic acid . The lactic acid ,8, but aoil sake ,10,11, dkuratsuki bacteria other than lactic acid bacteria exist during the sake brewing process, 46 bacterial isolates were collected at the early stage of the sake brewing process from Narimasa Sake Brewery in Toyama, Japan [Kocuria, 12 to Staphylococcus, 6 to Bacillus, 2 to Leifsonia, 2 to Microbacterium, and 1 to Enterococcus, inferred from the partial 16S rRNA gene sequences [Kocuria isolates have inhabited Narimasa Sake Brewery and are kuratsuki bacteria [Kocuria does not belong to the phylum Firmicutes but Actinobacteria [kuratsuki bacteria in the Narimasa Sake Brewery interact with S. cerevisiae and affect flavor of the sake products [To elucidate whether a, Japan . Among tbacteria . Kocuriabacteria . This waproducts . The bacproducts .Kocuria is specific to Narimasa Sake Brewery [Kocuria is not isolated from other sake breweries, so we elucidated the same in this study. In 2019, six bacterial isolates were collected from the sample at the late stage of the sake brewing process of Shiraki Tsunesuke Sake Brewery in Gifu, Japan. In this study, we isolated bacteria during the early stage of the sake brewing processes of this sake brewery, compared them, and then discussed them.Based on the DNA sequence data in sake,  Brewery . HoweverBacterial isolation was performed for four different sampling date samples from Shiraki Tsunesuke Sake Brewery . Three dThe V3–V4 region of the 16S rRNA gene from each single colony was amplified using region-specific PCR primers , designeBacillus isolates from Narimasa Sake Brewery [Bacillus species from DNA database  was spread on a TGY agar to count the number of colonies.http://www.R-project.org/).A chi-square test was performed between isolated numbers from the cultures at 15 °C and 30 °C using the software R (https://www.ncbi.nlm.nih.gov/), all 92 DNA sequences of bacterial isolates from Shiraki Tsunesuke Sake Brewery were found to be DNA sequences of the genus Bacillus. All partial 16S rRNA gene sequences are shown in We obtained 6 DNA sequences from the samples at the late stage of the sake brewing process in 2019, 28 at the early stage on 26 January 2021, 30 at the early stage on 28 January 2021, and 28 at the early stage on 9 February 2021 . Using tp > 0.05, chi-square test) between different sampling dates of cultures at 15 °C and 30 °C in each group and between different groups of cultures at 15 °C and 30 °C. An ethanol resistance test showed that isolate C1 belonging to Group 1 and isolate a12 belonging to Group 2 had an ethanol resistance to 20% ethanol  with 87% support of bootstrap , Staphylococcus (12 isolates), Bacillus (6 isolates), Leifsonia (2 isolates), Microbacterium (2 isolates), and Enterococcus (1 isolate) [kuratsuki Kocuria at the different species level [kuratsuki Bacillus at the different species level. Bacillus species have both positive and negative effects on drinks and foods [Although ronments , it was lococcus  isolateslococcus  isolateses level , leadingnd foods ,22,23,24Bacillus isolates of Groups 1 and 2 are kuratsuiki bacteria in Shiraki Tsunesuke Sake Brewery because these Bacillus isolates are specific to this sake brewery. In particular, the isolates of Group 1 are kuratsuki bacteria because this group includes the Bacillus strains that were isolated in 2019 at late sake production process, which has a high ethanol concentration (>15% of ethanol). Considering that the same Bacillus isolates were collected from the different samples with different sampling year, these isolates may have inhabited the Shiraki Tsunesuke Sake Brewery and entered the annual sake brewing process. The Bacillus isolates of Group 1 have a high ethanol tolerance and survive at the late stage of the sake brewing process. In contrast, Group 2 does not include the Bacillus isolates in 2019. This suggested that the Bacillus isolates of Group 2 might be less ethanol-tolerant compared with those of Group 1. However, the isolates of Group 2 had a high ethanol tolerance  that are managed by the Brewery Society of Japan. Each Kyokai yeast consists of a single lineage. It is possible that some Kyokai yeasts are not suitable for some sake brewery environments.It is important that  brewing  and thenuggested . KuratsuKuratsuki bacteria are not any contaminated bacteria, which are always entered in sake brewing. To our knowledge, although research on kuratsuki yeasts has been carried out, research on kuratsuki bacteria has not been performed except for lactic acid bacteria. We are changing the characteristics of sake and will produce a unique sake with different tastes and flavors by exchanging the kuratsuki bacteria from different sake breweries during the production process.Microorganisms play an important role in the production of flavor and taste, and influence the sake quality. Although bacteria die at the final stage of sake production, bacteria in sake are alive and grow temporarily during the sake production process.


The application of natural polymer matrices as medical device components or food packaging materials has gained a considerable popularity in recent years, this has occurred in response to the increasing plastic pollution hazard. Currently, constant progress is being made in designing two-component or three-component systems that combine natural materials which help to achieve a quality comparable to the purely synthetic counterparts. This study describes a green synthesis preparation of new bionanocomposites consisting of starch/chitosan/graphene oxide (GO), that possess improved biological activities; namely, good tolerability by human cells with concomitant antimicrobial activity. The structural and morphological properties of bionanocomposites were analyzed using the following techniques: dynamic light scattering, scanning and transmission electron microscopy, wettability and free surface energy determination, and Fourier transform infrared spectroscopy. The study confirmed the homogenous distribution of GO layers within the starch/chitosan matrix and their large particle size. The interactions among the components were stronger in thin films. Additionally, differential scanning calorimetry analysis, UV–vis spectroscopy, surface colour measurements, transparency, water content, solubility, and swelling degree of composites were also performed. The mechanical parameters, such as tensile strength and elongation at break (EAB) were measured in order to characterise the functional properties of obtained nanocomposites. The GO additive altered the thermal features of the composites and decreased their brightness. The EAB of composite was improved by the introduction of GO. Importantly, cell-based analyses revealed no toxic effect of the composites on HaCat keratinocytes and HepG2 hepatoma cells, although a pronounced bacteriostatic effect against various strains of pathogenic bacteria was observed. In conclusion, the starch/chitosan/GO nanocomposites reveal numerous useful physicochemical and biological features, which make them a promising alternative for purely synthetic materials. Nanotechnology has currently been attracting attention due to its appreciable role in the agricultural and food industries, where it is innovatively used to improve food quality and safety. The unique physical, chemical, and biological features of nanomaterials facilitate their application in medical and biological sciences, cosmetology, waste water treatment, and other fields ,2. In rePolysaccharides, due to the diversity of functional groups, are a particularly rewarding material for modification; furthermore, their low cost and abundance in natural resources makes them attractive for industrial applications. Moreover—being biodegradable, renewable, and most importantly nontoxic—polysaccharides are likely to successfully compete with synthetic polymers in the near future. Starch and chitosan belong to the biopolymers most frequently used in the production of sustainable food packaging. Chitosan is regarded as a biocompatible material, and its proven antimicrobial activity makes it suitable for medical, therapeutic, and pharmaceutical applications in drug delivery, tissue engineering, orthopaedic and periodontal applications, as well as wound healing ,4. ChitoA survey of the most recent literature indicates that graphene-containing bionanocomposites are very promising materials in the construction of biosensors, drug delivery systems, modified electrodes, energy-storage systems, and active packaging . Jamróz Drawing inspiration from all these innovative solutions we aimed to exploit the unique properties of potato starch and chitosan. We combined these two polymers into a matrix for the introduction of graphene oxide to obtain a bionanocomposites material with improved functional  and mechanical properties, which would hopefully broaden the range of possible applications, for instance in food packaging and preservation. According to European Union regulations, packaging and all other materials intended to have contact with food, need to be sufficiently inert not to release any exogenous substances in a quantity that could adversely affect human health, food composition, or its organoleptic characteristics . TherefoPotato starch (PS) was purchased from PPZ Bronisław company . This particular starch contained 19.90% moisture and 26.48% amylose. Chitosan (Ch) from shrimp shells was purchased from Sigma (CAS no 9012-76-4). Graphene oxide (GO) was purchased from NANOMATERIALS Leszek Stobiński . Glycerol—used as a plasticiser—was purchased from F.H.U. DOR-CHEM .The composites were made of starch and chitosan with the addition of graphene oxide. First, a 2% chitosan gel (Ch) was prepared by adding 30 g of chitosan to 1470 g of acetic acid solution (0.5%), stirred at 70 °C until a clear gel was obtained. Then a 4% starch gel (PS) was prepared by adding 30 g of potato starch into 720 g of distilled water with constant mixing at 80 °C for 30 min.Control Composite (Composite C)The control composite was prepared from starch and chitosan gels: 500 g of Ch gel was added to 250 g of PS gel and stirred approx. 30 min, until a homogeneous gel was obtained. Then 10 g of glycerol  and 25 g of distilled water were added. The mixture was stirred for 30 min and poured into plates to dry.Nanocomposite Iv/v) were added. Next, the mixture was stirred for 30 min and poured into plates to dry.Nanocomposite I consisted of PS/Ch with GO: 500 g of Ch gel and 250 g of PS gel were mixed together for 30 min. Then, 10 g of glycerol (a plasticiser) and 25 g of GO  and 12.5 g of H2O were added. The mixture was stirred for 30 min and poured into plates to dry. All samples . Zeta potential was calculated from the electrophoretic mobility of particles using the Smoluchowski model. The results are expressed as an average from three consecutive measurements with 20 runs.In our study we used the Drop Shape Analyzer Kruss DSA100M optical contact angle measuring instrument  for the evaluation of contact angles. The detailed methodology of experiments, as well as the surface free energy analysis, were presented in our previous paper . We used−1 using a MATTSON 3000 FT-IR  spectrophotometer. This instrument was equipped with a 30SPEC 30 Degree Reflectance adapter fitted with the MIRacle ATR accessory from PIKE Technologies Inc., Madison, Wisconsin, USA.The FTIR–ATR spectra of the composites were recorded in the range of 4000–7000 cmApproximately 4 mg of each composite sample was weighed and sealed into aluminium pans. Subsequently, the samples were heated from 25 °C to 400 °C at a rate of 10 °C/min. The empty pan was used as a reference. The tests were performed with the DSC 204F1 Phoenix differential scanning calorimeter . The parameters of the observed thermal transition were calculated with Proteus Analysis software . The analyses were performed in triplicate.The measurement of surface colour was carried out with the use of Konica MINOLTA CM-3500d equipment , with a 30 mm diameter window, using reference D65 illuminant/10° observer. The results were expressed using the CIELab system. The following parameters were determined: L* , a* - share of the green colour (a* < 0) or red (a* > 0), b*- share of blue (b* < 0) or yellow (b* > 0). The measurements were taken on a standard white background. The experiment was repeated five times.The thickness of composites was measured with a micrometer, catalog no. 805.1301 , with a 0.001 mm resolution. The sample thickness was the average of five measurements performed in various places within the gauge length area.600 is the absorbance at 600 nm and x is the film thickness (mm). A higher value of T indicates a lower degree of transparency.The transparency of composites was measured by exposing the film specimen to light absorption at a wavelength of 600 nm by the use of a UV–vis spectrophotometer (type Helios-Gamma 100-240). Rectangular film samples were placed directly into a spectrophotometer test cell and the empty test cell was used as a reference. The transparency (T) of the films was calculated by the equationThe UV–vis absorption spectra were recorded with a Shimadzu 2101 scanning spectrophotometer in the range of 200–800 nm using 10 mL, 10 mm-thick quartz cells.The morphology of as-prepared nanocomposites was studied using a high resolution JEOL JSM – 7500 F Field Emission Scanning Electron  equipped with a Transmission Electron detector (TED).2) and weighed in analytical balance obtaining initial weight of sample (M1). Then specimens were dried at 70 °C in an oven for 24 h, the initial dry mass (M2) was then analyzed gravimetrically. The samples were then dissolved in 30 mL of distilled water for 24 h at 25 °C. Afterwards the specimens were superficially dried using filter paper and weighed (M3). The residual film samples were dried at 70 °C for 24 h in an oven and the final dry mass was determined (M4). The analysis was performed in tetraplicate replications. Water content, solubility, and degree of swelling of films were calculated according to the equationsWater content, solubility, and the degree of swelling for films were determined according to the Souza et al.  procedur2 strips and put into holders. The initial grip separation between holders was 20 mm and the rate of grip separation was 2 mm/min. Tensile strength (TS) was calculated by dividing tensile force (maximum force at rupture of the film) by the cross-section area of the film. The percentage of elongation at the break (EAB) was calculated by dividing the elongation at rupture by the initial gauge length and multiplying by 100. The reported results were the average values of 10 replications.Dry composites were conditioned in desiccators at 25 °C and 52% relative humidity (RH) by using saturated solutions of magnesium nitrate-6-hydrate for 48 h prior to analysis. The samples for textural analysis were prepared according to ISO standards  and dete2 atmosphere.HepG2 human hepatocellular carcinoma cell line was bought from ATCC (#HB-8065) and were cultured in DMEM low glucose medium (Biowest) supplemented with 10% heat-inactivated fetal bovine serum , a mixture of antibiotics and antimycotics  and 2 mM glutamine . A spontaneously immortalised human keratinocyte cell line HaCat  was maintained in an RPMI 1640 medium  supplemented with antibiotics, 10% FBS and 2 mM glutamine as above. Both cell lines were kept at 37 °C with a 5% COThe HepG2 and HaCat cells were seeded on 96-well plates (2000 cells per well) in 100 μL of culture medium and left to attach for 24 h. The circular pieces of the tested composites, 6 mm in diameter (corresponding to the well size) were cut with a hole puncher, placed in sterile cell culture dishes and sterilised for 2 h under UV light. After sterilisation the composite circles were placed in the wells with the cells, then submerged in the medium. A non-treated control received no composites. After a 48 h incubation period they were removed and cell viability was determined using a CellTiter 96® Aqueous kit , which enables spectrophotometric monitoring of MTS tetrazolium reduction by viable cells. Two independent experiments were performed, each in tetraplicate.-Staphylococcus aureus ATCC 25923-Pseudomonas aeruginosa ATCC 27853-Enterococcus faecalis ATCC 29212-Escherichia coli ATCC 8739-Proteus mirabilis NCTC 11938A study was carried out on the strains from the ATCC and NCTC collections:3 were poured into sterile plastic Petri dishes with a diameter of 90 mm. After solidification of the MH medium, 200 μL of bacterial suspensions were spread on the surface with a sterile swab. The circular pieces of tested film (0.5 cm diameter) were placed on the culture surface. Three discs were placed on each Petri dish. Petri dishes were incubated 48 h in 37 °C, aerobic atmosphere. After 48 h incubation, a measurement of the inhibition zone of growth was made. Three independent experiments were performed for all the samples.Bacterial suspensions with an optical density of 0.5 McFarland were used. The Mueller–Hinton (MH) medium  with a volume of 16 cmp = 0.05, using Statistica v. 8.0 software . A Fisher test was used for the determination of statistically significant differences.The experimental data was subjected to an analysis of variance, at the confidence level of All the tested composites possessed a positive zeta potential in the range between ca. 28 mV and 52 mV . The zetThe analysis of particle size is more complicated. Particle size appeared to depend on composite thickness. The largest particles, about 2000–2500 nm, were observed in the thin composites, while in the thick samples, the particle size was about 600–900 nm.Regardless of the concentration, the addition of GO increased the hydrophilic properties of the thin samples  bending. The peak near 1655 cm−1 probably represented the carbonyl group stretching (amide I). In the starch spectrum, the presence of the broad band at 3350 cm−1 was associated with OH stretching. The multiple bands between 950–1150 cm−1(−1), the asymmetric vibrations of the ring (around 1100 cm−1) and stretching vibrations of the C-O bonds (the range of 960–1080 cm−1). The spectra of both samples (starch and chitosan) revealed multiple bands at 2916–2936, 2855, 1405–1465, and at 1245 cm−1 which come from the -CH2- group, as well as at 2880–2900 and 3200 cm−1 bands, which represented the C-H groups within the polysaccharide molecules. The spectrum of chitosan/starch composite film showed that the addition of starch caused the shift of the chitosan amino peak from 1578–1584 cm−1. This result indicated the interactions between the hydroxyl groups of starch and the amino groups of chitosan. The peak of the hydroxyl groups could not be used to evaluate the interactions because of the masking effects of glycerol content [FTIR spectroscopy was used to examine the interactions between chitosan and starch a. The pr1150 cm−1a corresp−1, the C-O (epoxy) stretching vibration peak at 1227 cm−1, the C-O  stretching vibration peak at 1065 cm−1, and the vibration and deformation peaks of O-H groups at 3412 cm−1 and 1627 cm−1 respectively [The FTIR spectra of PS/Ch composite (control sample) and GO nanocomposites b displayThe DSC curves  demonstrThe estimated enthalpy change (ΔH) of melting for nanocomposite I was significantly higher than in the other samples . These rDetailed colour and transparency analysis of the composites confirmed their high transparency and pale yellow hue , Table 5The values of the L* parameter remained high for both the control and nanocomposite samples, this indicated brightness . The incThe GO UV–vis absorption spectrum presented a characteristically sharp absorption peak at about 233 nm and a broad shoulder at 290 nm–305 nm . The peaThe morphology of chitosan/starch-based nanocomposites containing graphene oxide (GO) is shown in The water content in all the composites does not vary much and ranges from 8.81% to 10.92% . A desceThe solubility of the thick samples  than their thicker counterparts. The thin films were comprised of larger particles (Tab. 1). We speculate that much stronger intermolecular forces exist in thin composites, this is what makes them much more resistant to breakage. The DLS results confirm that in the thick films, the interactions among GO, S, and Ch are much weaker: the particle size is smaller after dissolution and the whole system is less dense.Tensile strength and elongation at break of composites are important parameters for assessing the ability to maintain the integrity of the composites in the presence of environmental stress factors. Therefore, these parameters determine the particular application of such nanomaterials in packaging and other industries . High te2, CO2) flow and exchange by the film pieces that covered the cultures.The cell viability assays demonstrated that incubation with graphene nanocomposites is well tolerated by human skin keratinocytes. A slowdown of the cell proliferation rate was noticed, but a significantly lower number of viable cells as compared to the non-treated control was observed only for nanocomposite II . Human lThis lack of cytotoxicity is very important, because numerous previous studies reported a considerable level of cytotoxicity in various cell lines . One of Escherichia coli, Proteus mirabilis, and Staphylococcus aureus cultures, where the growth inhibition zone had 5 mm diameter. The growth inhibition was independent of the graphene concentration. In the case of Enterococcus faecalis the growth inhibition was stronger for the samples with lower graphene concentration (0.3 mm) as compared to the control. In the Pseudomonas aeruginosa cultures, the GO content did not enhance bactericidal effect, nevertheless, bacterial growth inhibition in the presence of all the composites was clearly visible (The bacteriostatic activity of the graphene-containing nanocomposites has been confirmed in  visible . This inEscherichia coli, Proteus mirabilis, and Staphylococcus aureus, but importantly, the lack of toxicity towards human cells was confirmed, which is a great advantage and broadens the range of possible applications in various branches of industry. Collectively, the obtained results indicate the potential application of these nanocomposites as active packaging materials. Further experiments could be designed to test these materials for the storage of various food products.Here we have described a green synthesis preparation of new bionanocomposites, enriched with GO nanoparticles and based on binary starch/chitosan matrix. We checked how graphene content and film thickness affected their physicochemical and mechanical parameters. Electron microscopy SEM and TEM analysis on the nanocomposites illustrated an even distribution of GO layers within the polysaccharide matrix, which indicated homogenous GO distribution and lack of GO aggregates. The particle size measurements performed with DLS technique confirmed that thin composites had more than double the particle size of their thick counterparts. The higher GO content led to the decrease in zeta potential values and increased EAB values, which indicates improved elasticity. The DSC analysis confirmed the effect of GO on the thermal characteristics of nanocomposites as well as the higher melting temperatures determined in the GO containing samples. Incorporating graphene into S/Ch matrix slightly altered film colours, but the samples were still transparent. All the tested composites were highly permeable regardless of the GO content, which is an advantage in the aspect of biodegradability, although it may limit certain applications. The mechanical studies revealed that thin films were more resistant to rupture. Both thick and thin films showed high elasticity/expandability. The introduction of GO considerably improved nanocomposite elongation at break. The GO-containing nanocomposites exhibited bacteriostatic activity against


Temperature also increased development rate, but there was no effect of ploidy level. We did find interactive effects between ploidy and temperature for gene expression, body size and swimming performance, confirming that the consequences of cell size are temperature dependent. Triploids with larger cells performed best at cool conditions, while diploids performed better at warmer conditions. These results suggest different selection pressures on ectotherms and their cell size in cold and warm habitats.Cell size may be important in understanding the thermal biology of ectotherms, as the regulation and consequences of cell size appear to be temperature dependent. Using a recently developed model system of triploid zebrafish (which have around 1.5-fold larger cells than their diploid counterparts) we examine the effects of cell size on gene expression, growth, development and swimming performance in zebrafish larvae at different temperatures. Both temperature and ploidy affected the expression of genes related to metabolic processes ( Cell size is emerging as an important trait in understanding the thermal biology of ectothermic animals, as the regulation and consequences of cell size appear to be dependent on temperature . The preDaphnia pulex mature faster than their diploid conspecifics, but only at low temperatures  with a larger genome and especially polyploids suggests that organisms with a larger genome perform better in colder environments : Polyploeratures . Fish speratures . Laborateratures . In addieratures . As cellA role for oxygen in shaping the thermal response in growth is likely . Oxygen Cell size may also affect oxygen supply, as oxygen diffuses more easily across lipid membranes than through aqueous cytosol . TherefoThis study examined the effects of cell size on gene expression, growth, development and swimming performance in zebrafish larvae reared at different temperatures. We used a triploid zebrafish model that we developed in a previous study . We demoMaintenance of adult zebrafish and triploidy induction have been described in detail in in vitro fertilization. Triploidy was induced by using a cold shock treatment; 3 min after fertilization the collected eggs were immersed in 4°C E2 medium  for 20 min. Eggs were fertilized with a pooled sperm solution of 8–12 males to ensure a heterogeneous offspring. In addition, eggs of different females were randomly distributed over the experimental treatments. Different batches of eggs were harvested from multiple rounds of fertilization, alternating between different parental tanks with similar genetic background. We did not divide the egg batches in half to produce diploids and triploids, due to the vulnerability of the eggs right after fertilization. However, we do not expect family effects as we obtained diploids and triploids from similar parental tanks, although not in the same round of fertilization. Multiple batches of eggs were reared for each experiment: seven for genome size and DNA condensation, two for gene expression, four for development rates, and for growth and swimming performance six batches were reared.Eggs were collected by both natural spawning in mass breeding tanks and 1, the European guidelines for animal experiments (Directive 2010/63/EU)2 and institutional regulations.All experiments were carried out in accordance with the Dutch Animals Act–5% methylene blue). During development, these tanks were constantly aerated and maintained in water baths connected to a circulating heating/cooling system (Grant LT ecocool 150) at the aforementioned temperatures ±0.5°C. Dead embryos or larvae were removed from the setup daily. Survival was affected by ploidy level, but not rearing temperature [see also Within 2 h after fertilization, diploid and cold shocked embryos were divided over three temperature treatments: 23.5°C, 26.5° (control temperature), and 29.5°C. The embryos were kept in 48-wells plates  with a mesh bottom and placed in a rearing tank containing E3 medium  and larvae at 29.5°C were reared until 4 dpf, at which point they reached the same developmental stage as 26.5°C larvae reared until 5 dpf. At this time, larvae have almost fully resorbed their yolk sack and are able to produce a proper escape response. Throughout the manuscript, when we indicate 5 dpf, we refer to the developmental stage of 5 dpf for larvae reared at the three different temperatures.Triploidy induction was verified in cold shocked larvae at 5 dpf using a propidium iodide (PI) staining to quantify the amount of nuclear DNA. This method has been described in detail in In addition to triploidy verification, we also compared the PI staining within diploid and triploid larvae reared at different temperatures. Therefore, we also measured samples containing two diploid larvae for each temperature treatment. Lastly, we calculated the G2/G1 ratio of the cells for each condition, which is a measure for the dividing potential of the cells. The R package flowPloidy  was used2O for qPCR.Diploid and cold shocked larvae of all three temperature treatments were collected for qPCR analysis at 5 dpf. Two or three larvae were pooled in 2 mL Eppendorf tubes and immediately frozen in liquid nitrogen, with at least five replicate samples per treatment. Details about the qPCR preparation can be found in rps11, previously known as 40S); actin, beta 1 (actb1); eukaryotic translation elongation factor 1 alpha 1, like 1 (eef1a1| 1); polymerase (RNA) II (DNA directed) polypeptide D (polr2d); ribosomal protein L13a (rpl13a) and TATA box binding protein (tbp). These housekeeping genes are frequently used to normalize gene expression of genes of interest, as they are involved in a range of basic cellular processes. The genes of interest are: citrate synthase, mitochondrial (cs); L-lactate dehydrogenase A chain (ldha); L-lactate dehydrogenase B-A chain (ldhba) and heat shock cognate 70-kd protein, tandem duplicate 1 (hsp70.1). Primer sequences and the involvement of these genes in cellular and physiological processes can be found in The expression levels of six housekeeping genes and four genes related to metabolic processes and temperature responses were analyzed: ribosomal protein S11 , by determining their developmental stage according to 3.At each time point, five diploid and five cold shocked embryos were staged of all temperature treatments. In our final analysis, we only included development of embryos that morphologically appeared to have developed normally at 5 dpf . Length was measured at the developmental stage of 5 dpf (protruding mouth stage), using pictures of larvae taken with a Leica MZ FLIII stereomicroscope equipped with a Leica DFC450 C camera and the segmented line tool of the ImageJ programMaximum swimming velocity of diploid and cold shocked larvae of the three rearing temperatures was assessed at 5 dpf using a DanioVision system . Details of the procedure can be found in post hoc test was performed to compare between the different ploidy and temperature groups.The collected data were analyzed using RStudio version 1.1.383, respecting a significance threshold of α = 0.05. To compare genome sizes and to test for the effect of rearing temperature on DNA condensation for diploid and triploid larvae, we used a general linear model and subsequent ANOVA. Effects of ploidy and rearing temperature and the interaction between these factors on the ratio of cells in the G2 and G1 phase were also tested with ANOVA. Tukey’s post hoc test was used to annotated differences between ploidy levels and temperature groups.Gene expression data were analyzed for each gene separately, using a general linear model of which the frequency distribution of the residuals was visually checked for normality. We used a subsequent ANOVA to test for the effects of ploidy level and rearing temperature and the interaction thereof. Tukey’s post hoc test to compare between the different ploidy and temperature groups.Development rates of diploid and triploid larvae reared at different temperatures were compared using a general linear model, after visually checking the frequency distribution of the residuals as being normally distributed. We tested for the effects of ploidy level, rearing temperature, development time and the interaction of these factors. Within temperature treatments we tested for the effect of ploidy level, again using general linear models and subsequent ANOVA. Length measurements were analyzed using a general linear model to test for the effects of ploidy level, rearing temperature and the interaction of these factors, as the residuals were normally distributed. We used ANOVA and a subsequent Tukey’s For the swimming performance data, we first calculated the proportion of diploid and triploid responders vs. non-responders for each temperature treatment and stimulus number, as we observed a bimodality in startle responses . A lineaF1,59 = 650.18, p < 0.001, F1,59 = 9.57, p = 0.003, Flow cytometry clearly distinguished between cells in the G1 phase and cells in the G2 phase , with flF1,58 = 46.32, p < 0.001, The ratio between G2 and G1 phase cells reflects how many cells are in the process of dividing. At the lowest rearing temperature, we found a higher G2/G1 ratio in diploids compared to triploids, but at the highest temperature this pattern was reversed, reflecting a significant interaction between ploidy and rearing temperature  and one based on only those genes whose expression did not vary with ploidy and temperature , indicating that the stimulating effect of warm conditions on development was similar for diploids and triploids.Both diploid and triploid larvae progressed faster through the different developmental stages at higher temperatures . This pattern seemed to be reversed at an acute low temperature, although the effect of ploidy was not significant  triploids are also reported to maintain higher routine metabolic rate at low temperatures compared with diploids  (Growth and size attained is another important performance metric . Warmer ulentus) . Indeed,ulentus) . In contulentus) . A stronulentus) . Similarulentus)  and exhiulentus) .eef1a1| 1 and rpl13 with higher rearing temperature, both of which are involved in protein synthesis during translation. Although expression levels of genes cannot be directly interpreted as different amounts of protein product from these genes, due to differences in translation and post-translational modifications, the higher expression levels would suggest increased protein synthesis to fuel the faster growth and development at higher temperatures. Interestingly, gene expression of polr2d, coding for RNA polymerase II, and tbp, coding for TATA box binding protein, were higher at lower temperatures. Possibly, transcription, rather than translation was a rate limiting step in colder temperatures and the higher gene expression helps to maintain transcription and thus enhance cell growth even at lower temperatures (tbp), perhaps compensating for their lower DNA content. Moreover, fluorescence intensity of stained DNA decreased with temperature, suggesting that DNA might be more condensed at lower temperatures. It is possible that the DNA configuration of the larvae at 29.5°C is easier accessible for PI, which is a small molecule, due to more flexible bended DNA .All data files will be made available in the DANS EASY archive (Ethical review and approval was not required for the animal study because the experiments were performed with larvae up to a developmental stage of 5 days post-fertilization, a stage where larvae are not yet dependent on external feeding.IP and WV conceived the experimental design and analyzed the data. IP performed the experiments and wrote the first draft of the manuscript. AH participated in collecting data on development rates. WV and AH edited the manuscript. All authors contributed to the article and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood RNA-sequencing analysis of the prenatal human brain at different stages of development shows that areal transcriptional signatures are dynamic and coexist with developmental and cell-type signatures. It is also crucial for unravelling the pathology of neurodevelopmental and neuropsychiatric disorders that often preferentially affect specific brain regions and/or neocortical areas4. Early patterning of the developing telencephalon is orchestrated by morphogenetic gradients of growth factors including bone morphogenetic proteins, Wnts, sonic hedgehog and, most prominent in the cortex, fibroblast growth factor5. However, the molecular patterns that arise as a result of these gradients are less well understood.Understanding when brain regions acquire their unique features and how this specification occurs has broad implications for the study of human brain evolution, including species-specific developmental differences that may have contributed to the expansion of cortical areas such as the prefrontal cortex (PFC)6. We sampled cells from 10 distinct major forebrain, midbrain and hindbrain regions from 13 individuals 7, ZIC2 9 and NRP1 10  of clusters were strongly enriched for a single or related brain region  and V1 are transcriptionally distinct from each other1, denser sampling of areas between the ALM and V1 reveals a gradient-like transition between cell-type profiles19. We sought to expand upon these findings by profiling single cells from distinct cortical areas, yielding 387,141 high-quality cells, after filtering (Methods) , IPCs and excitatory neurons) across cortical areas. We validated cortical area sub-dissections by quantifying the expression of nes Fig. .Fig. 3Co17, the specificity of neuronal areal markers was significantly higher compared with RG , a putative driver of brain expansion involved in neuron differentiation and cortical migration27. In the PFC, area-specific transcription factors include HMGB2 and HMGB3, which are differentially expressed by neural stem cells at distinct stages of development28 and are thought to be key regulators of differentiation. Of note, HMGB3 mutations can result in severe microcephaly. We also found upregulation of the genes encoding the transcription factors NEUROG1 and NEUROG2 in PFC neurons. Although these PFC-specific genes have been previously described as regulators of neuronal differentiation, they have not been implicated or studied in the process of cortical arealization.Within each set of areal marker genes, we identified genes encoding transcription factors that were robustly enriched in cells of a specific area as well as transcription factors with a broad frontal or caudal enrichment Fig. . A subse20, we observed that while area-specific gene signatures are composed of significant and specific marker genes, they also change substantially throughout this period  to identify the ventricular zone and cortical plate  Fig. . We quanate Fig. , 9. We cs18 Fig. . These sNEFL, SERPINI1 and NR4A2. All three markers showed largely equal intensity levels across cells in PFC, somatosensory, temporal, and V1 cortex, but their relative spatial distribution changed substantially  is a strong determinant of gene-expression signature composition. Together, these observations suggest that the dynamics of area-specific gene-expression signatures are surprisingly fast moving and cell-type-specific  at the University of California, San Francisco. Two sets of samples included twins: GW20_31 and GW20_34; GW22 and GW22T.Brain dissections were performed under a stereoscope with regards to major sulci to identify cortical regions. Of note, all dissections were performed by the same individual (T.J.N.) to enable reproducibility and comparison between samples. Tissue was incubated in 4 ml of papain/DNAse solution (Worthington) for 20 min at 37 °C, after which it was carefully triturated with a glass pipette, filtered through a 40-µm cell strainer and washed with HBSS. The GW22 and GW25 samples were additionally passed through an ovomucoid gradient (Worthington) in order to minimize myelin debris in the captures. The final single-cell suspension was loaded onto a droplet-based library prep platform Chromium (10X Genomics) according to the manufacturer’s instructions. Version 2 was used for all samples except for GW19_2, GW16, and GW18_2 for which version 3 chemistry was used. cDNA libraries were quantified using an Agilent 2100 Bioanalyzer and sequenced with an Illumina NovaSeq S4.29 for each individual capture lane, then required at least 750 genes per cell and removed cells with high levels (>10%) of mitochondrial gene content. These strict metrics for quality control preserved no more than 40% of cells for downstream analysis, and re-analysis of the data for specific brain structures or cell types may benefit from less stringent QC for additional discovery. Our goal was to obtain clean populations with a high validation rate for a better understanding of arealization signatures. The resulting ~700,000 cells passing all thresholds were used in downstream analyses.We filtered cells using highly stringent quality control (QC) metrics. In brief, we discarded potential doublets using the R package scrubletWe used a recursive clustering workflow to understand the cell types present in our dataset. In order to minimize potential batch effects and to increase detection sensitivity of potential rare cell populations, we performed Louvain–Jaccard clustering on each individual sample first. After initial cell type classification, we sub-clustered all the cells belonging to a cell type to generate the most granular cell subtypes possible. We then correlated subtypes between individuals based upon the gene scores in all marker genes to bridge any batch effects, and iteratively combined clusters across all individuals and cell types. For this study, we combined the clusters within a single cell type across all individuals once, and again with all clusters from all individuals and cell types, resulting in two iterative combinations. The annotations at each step are preserved in the supplementary tables to enable reconstruction at any point in the pipeline.https://software.broadinstitute.org/morpheus) across all rows and columns using one minus the Pearson correlation for the distance metric.Cluster hierarchies are generated from matrices correlating all clusters to one another using Pearson’s correlation in the space of gene scores for all marker genes across all groups. Hierarchical clustering is performed within Morpheus , then determining, at each cluster, the fraction of neighbours belonging to a different cluster. An edge is drawn between 2 nodes if >5% of nearest neighbours belong to the opposite cluster in at least one of them. An edge’s width at the node is proportional to the fraction of nearest neighbours belonging to the opposite node, with the maximum fraction of out-of-node neighbours across all clusters represented as an edge width of 100% and equal to node width. The full code adaptation and implementation of this analysis is described in the function buildConstellationPlot in this paper’s associated Github repository.To visualize and quantify the global relationships and connectedness between cell types, cell type subclusters, or cell type-area groups, we implemented the constellation plots described in ref. Constellation plots were quantified by using a summary of the input values described above. For each cell type or area connection, the number of edges between two groups was multiplied by the average fraction of cells meeting the threshold for a connection within the group. This resulting number was called the connectivity index.30. Scores were generated for each set of up to 10,000 randomly subsetted cells from the group using the function moduleEigengene function, Scores were calculated based on the intersection of the gene set of interest and genes expressed in the subset of cells. For the area-specific signatures, differential expression was performed as described above, and the gene signatures from late stage neurons across all areas were used to calculate module eigengenes for the radial glia and IPC populations.Module eigengenes were calculated for numerous gene sets using the the R package WGCNAP-value cut-off of 0.05. Adjusted P-values were based on Bonferroni correction using all features in the dataset. We performed this step separately for each cell type and each individual, since we observed that gene specificity was highly dynamic throughout the developmental process. We then combined the individual gene lists of each cell type and area, and annotated the stage(s) at which each gene appeared to be specific. We binned individuals into three stages: early , middle  and late (GW22 and GW25). We ranked upregulated genes by specificity by calculating their gene score, which we defined as the result of a gene’s average log fold-change × enrichment ratio, in turn defined as the percentage of cells expressing the gene in the cluster of interest divide by the percentage of cells expressing in the complement of all cells. Dot plots used to visualize the expression of distinct marker genes across cell types and/or cortical areas were generated the custom function makeDotPlot available in our code repository, which makes use of the Seurat function DotPlot. In brief, for each gene, the average expression value of all non-zero cells from each group  is scaled using the base R function scale, yielding z-scores. Scaling is done to enable the visualization of genes across vastly different expression ranges on the same colour scale.The expression profiles of cells from each subcluster or cortical area were compared to those of all other cells using the two-sided Wilcoxon rank-sum test for differential gene expression implemented by the function FindAllMarkers in the R package Seurat and selected based on an adjusted 31 and downloaded from the transcription factor database AnimalTFDB332, available at http://bioinfo.life.hust.edu.cn/static/AnimalTFDB3/download/Homo_sapiens_TF.Areally enriched marker genes obtained as described above were annotated against a comprehensive list of 1,632 human transcription factors described inTo quantify the degree of (dis)similarity of molecular signatures across distinct cell types, cortical areas, and/or developmental stages, we calculated the overlap between all sets of cell type and area-specific gene markers at each stage, and visualized these comparisons using Sankey diagrams using the function ggSankey from the ggvis R package. We then calculated the proportion of genes for each node shared with every other node, and clustered nodes hierarchically by calculating their euclidean distances based on their proportions of shared genes. The code used to construct the overlap matrices, create the plots and quantify the results is described in the functions buildSankey and buildHeatmap in our Github repository.22 and scVelo v0.2.223. Reads that passed quality control after clustering were used as input for the Velocyto command line implementation. The human expressed repeat annotation file was retrieved from the UCSC genome browser. The genome annotation file used was provided by CellRanger. The output loom files were merged and used in scVelo to estimate velocity. For the combined cortical analysis, cells underwent randomized subsampling (fraction = 0.5), and were filtered based on the following parameters: minimum total counts = 200, minimum spliced counts = 20 and minimum unspliced counts = 10. The final processed object generated a new UMI count matrix of 18,970 genes across 195,775 cells, for which the velocity embedding was estimated using the stochastic model. The embedding was visualized using UMAP of dimension reduction. The velocity genes were matched by cortical area and were estimated using the rank velocity genes function in scVelo. Computational analysis of the transcriptomic data described in detail above were performed using R 4.024 and Python 3, the R packages Seurat (version 2 and version 3)26, googleVis27, dplyr and ggplot228, the Python packages Velocyto v0.1722 and scVelo v0.2.223 as well as the custom-built R functions described. Our reproducible code is available in the Github repository associated with this manuscript.Velocity estimates were calculated using the Python 3 packages Velocyto v0.17SOX2 was chosen to mark radial glia, EOMES was chosen to mark IPCs, and BCL11B and SATB2 were chosen to marker excitatory neuronal populations with previously validated changing co-expression patterns. POLR2A was used as a positive control for the technology. The remaining genes were selected based upon their status as a specific marker gene for excitatory neuron clusters of interest.Marker genes for validation with the spatial omics platform were chosen first by identify useful cell type markers within the dataset. 33. Samples were then mounted to APS-coated coverslips, and fixed for 10 min in 4% PFA. Samples were then washed with PBS, and processed for spatial analysis. The spatially resolved, multiplexed in situ RNA detection and analysis was performed using the automated Rebus Esper spatial omics platform (Rebus Biosystems). The system integrates synthetic aperture optics (SAO) microscopy34, fluidics and image processing software and was used in conjunction with smFISH chemistry. Individual transcripts from target genes were automatically detected, counted, and assigned to individual cells, generating a cell × feature matrix that contains gene-expression and spatial location data for each individual cell, as well as registered imaging data, as follows.Samples for spatial transcriptomics were dissected from primary tissue as described above. Samples were flash frozen in OCT following the protocol described in the osmFISH protocol33. Fresh frozen brain tissue sections (10 µm) were cut on a cryostat, mounted on the treated coverslips and fixed for 10 min with 4% paraformaldehyde  in PBS at room temperature, rinsed twice with PBS at room temperature and stored in 70% ethanol at 4 °C before use. The sample section on the coverslip was assembled into a flow cell, which was then loaded onto the instrument. The hybridization cycles and imaging were done automatically under the instrumental control software. In brief, primary probes for all target genes were initially hybridized for 6 h and probes not specifically bound were washed away. Readout probes labelled with Atto532, Atto594 and Atto647N dyes for the first 3 genes were then hybridized, washed, counterstained with DAPI and then imaged with an Andor sCMOS camera  through a 20×, 0.45 NA dry lens  with a 365-nm LED for DAPI and 532-nm, 595-nm and 647-nm lasers configured for SAO imaging. Multiple fields of view (FOVs) were imaged for each channel within the region of interest (ROI). Single z-planes with 2.8 µm depth of field were acquired for each FOV. After imaging, the first three readout probes were stripped and the readout probes for the next three genes were then hybridized, imaged and stripped. This process was repeated until readout was completed for all genes.Rebus Biosystems proprietary software was used to design primary target probes (22–96 oligonucleotides) and corresponding unique readout probes (assigned and labelled with Atto dyes) for each gene. The oligonucleotides were purchased from Integrated DNA Technologies and resuspended at 100 µm in TE buffer. Coverslips  no. 1152460, Azer Scientific) were functionalized as previously publishedxy positions and signal intensities. Nuclei segmentation software based on StarDist35 identified individual cells by finding nuclear boundaries from DAPI images. The detected RNA spots were then assigned to each cell using maximum distance thresholds. The resulting cell × feature matrix contains gene counts per cell along with annotations for cell location and nuclear size.Using the Rebus Esper image processing software, the raw images were reconstructed to generate high-resolution images . RNA spots were automatically detected to generate high fidelity RNA spot tables containing Kernel density estimation plots were created from individual gene spot location maps retrieved from the spatial transcriptomics pipeline. They were created using the seaborn kdeplot function in Python with shading and cmap colouring. They were merged together for Fig. SOX2, EOMES and DLX6) were removed from the analysis. Interactions of 0.05 or more were preserved and visualized with Cytoscape v3.8.2 using a force-directed biolayout. Individual nodes were coloured by their colour in the merged image file in Fig. Using the cell × feature matrices, we eliminated all spots with less than ten counts for signal. Pearson’s correlations were then performed across the genes within each dataset and filtered for self-correlation. Positive control (POL2RA) and non-excitatory neuron cell type markers (Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03910-8.Supplementary InformationThis file contains the full descriptions for Supplementary Tables 1–16.Reporting SummarySupplementary TablesSupplementary Tables 1–16.


Acute fatty liver of pregnancy (AFLP) is an acute, rare and potentially lethal disease typically occurring in the third trimester of pregnancy. So far, there is no effective means of prevention. Therefore, in this study, we retrospectively analyzed the clinical features of AFLP patients for a better understanding. Meanwhile, for the first time, the genetic background associated with the onset of AFLP was discussed by high-throughput sequencing, hoping to provide evidence for genetic counseling and prenatal diagnosis of AFLP.Thirteen AFLP patients admitted to our hospital and other hospital from March 2012 to February 2020 were selected. Clinical data about general condition, laboratory test, liver biopsy and the prognosis of mother and fetus were collected for retrospective analysis. In addition, the peripheral blood of five patients with AFLP and one newborn infant of his mother with AFLP was sequenced with whole-exome sequencing and gene mutation was analyzed by bioinformatics methods.The initial symptoms of AFLP varied differently, with jaundice , fatigue  and nausea and vomiting  being the most common. Moreover, the main maternal complications were coagulopathy , followed by acute renal dysfunction . Raised serum bilirubin, transaminases and uric acid were found in all patients (100%), hypoglycemia was found in six patients (46%) and fatty liver on ultrasound was seen in five patients . One (7%) maternal death occurred and all neonates survived delivery. In addition, to our surprise, whole-exome sequencing showed that no gene mutation in related enzymes involved in fatty acid metabolism was noted in the pregnant women and children receiving genetic testing.Early visit, early detection, early termination of pregnancy and multidisciplinary comprehensive treatment are the key factors to improve the prognosis of AFLP patients and their newborn infants. Furthermore, although limited size of study, to our knowledge, this report is the first to present the lack of common mutation involved in fatty acid oxidation in Chinese patients with AFLP via whole-exome sequencing. Thus, further studies are needed with larger and more varied samples to validate the conclusion.The online version contains supplementary material available at 10.1186/s12884-021-04095-8. Acute fatty liver of pregnancy (AFLP) is a relatively rare but potentially fatal obstetric emergency. Epidemiological studies provide the incidence for AFLP of 1 per 7000 to 15,000 deliveries . With adThirteen AFLP patients admitted to Mengchao Hepatobiliary Hospital, Fujian Medical University and 900th Hospital of PLA Joint Logistics Support Force from March 2012 to February 2020 were selected. This study was approved by the Research Ethics Committee of Mengchao Hepatobiliary Hospital, Fujian Medical University and 900th Hospital of PLA. Informed consent was obtained from all individual participants included in the study.9/L; (9) ascites; (10) elevated transaminase (> 42 U/L); (11) elevated ammonia (> 47 μmol/L); (12) acute renal injury (Cr > 150 μmol/L); (13) coagulopathy ; (14) “bright liver” on ultrasound; (15) microvesicular steatosis on liver biopsy.Patients were enrolled in the study if they met the Swansea criteria, a diagnostic tool for AFLP , 2. AfteThe general information, clinical manifestations, laboratory indicators, abdominal ultrasound, mode of delivery, fetal conditions, treatment measures, length of hospital stay, and maternal and infant prognosis of 13 AFLP patients were analyzed retrospectively and detailly recorded in Additional file Percutaneous hepatic biopsy was performed under the guidance of ultrasonography on postnatal day 16 or day 23. Liver biopsy specimens were fixed with 10% neutral formaldehyde, paraffin-embedded, and sliced into 4-μm-thick sections. Histological evaluation was performed according to the conventional protocol by two pathologists blinded to the experiment.Whole-exome sequencing (WES) was performed by Running Gene Inc.  to discover the causal gene. Peripheral blood samples from 5 probands and 1 kid were collected. According to the manufacturer’s instructions, DNA samples were isolated using a DNA Isolation Kit , qualified by a Qubit dsDNA HS Assay Kit , fragmented into 200–300 bp length by Covaris Acoustic System  and then prepared with a KAPA Library Preparation Kit . Hybridization of the prepared libraries to the capture probes and removal of non-hybridized DNA fragments were carried out according to the Agilent SureSelectXT2 Target Enrichment System . The final products were sequenced as paired-end 150-bp reads on the Illumina HiSeq X platform. The mutations that affected the encoding of amino acids in the exonic regions or in the splice sites including missense, frameshift, stoploss, stopgain and splicing variants were filtered and annotated using GATK variant filtration and ANNOVAR in additional file Statistical analysis was conducted using SPSS software version 17.0. Data in normal distribution were displayed as mean ± standard deviation (SD). The counting data were expressed as percentage or rate.A retrospective analysis was performed on 13 patients with final clinical diagnosis of AFLP admitted from March 2012 to February 2020, ranging from 23 years old to 39 years old, with an average age of 29.2 years. There were 5 cases of multipara and 8 cases of primipara. The onset time ranged from 32 to 38 + 6 weeks, with an average time of 34 + 5 weeks. Among them, there were 4 cases (31%) with gestational weeks less than 34 weeks, 7 cases (54%) with 34 ~ 36 + 6 weeks, and 2 cases (15%) with 37 weeks or more. Visibly, this kind of disease is concentrated in the third trimester. The patients’ visit time is generally 0–30 days after the onset and the diagnosis time is 0–2 days after the visit. All pregnancies were terminated within 24 h of definitive diagnosis. Two pregnant women (case 1 and case 2) were accompanied by serious coagulopathy and DIC due to delayed care seeking, among which 1 pregnant woman died. There were 11 cases of cesarean section and 2 cases of vaginal delivery. Above details are given in Table Among the 13 patients, the initial symptoms of AFLP varied differently, with jaundice , fatigue 8/13, 62%) and nausea and vomiting  being the most common. Besides, three pregnant women (23%) were combined with HELLP syndrome or preeclampsia  maternal death occurred and all neonates 100%) survived delivery. The newborns delivered were all single births, including 8 boys, 5 girls, 10 premature infants and 3 full-term infants  underwent liver biopsy guided by B-ultrasound 16 days and 23 days postpartum during the recovery period, respectively. The main pathological feature is characterized by microvesicular hepatic steatosis in Fig. Many foreign studies have pointed out that the development of AFLP is related to genetic mutations in pregnant women or newborns. Mutations reported so far are common defects in enzymes involved in fatty acid oxidation, such as LCHAD, CPTl, MCAD or SCAD , 10. ComPrevious researches have shown that AFLP typically occurs during the third trimester of pregnancy, especially after 30 weeks of gestation. Also, there are rare cases occurring as early as 22 weeks of gestation –13. ConsIn our study, the main symptoms included jaundice, nausea, vomiting and abdominal pain in all pregnant women. However, all these manifestations were nonspecific. Patients were mainly hospitalized for further treatment about abnormal liver function. It is estimated that up to 3% of all pregnancies are complicated by liver disorders, which is broadly divided into 3 different groups: co-incident liver disease , pre-existing liver disease , and diseases unique to pregnancy . A timelTo terminate the pregnancy as early as possible is the utmost importance for the treatment of AFLP, in which cesarean delivery is the first choice of mode of delivery. Delayed termination of pregnancy caused by missed diagnosis, misdiagnosis, or conservative treatment may lead to rapid progression of the disease, which could make the condition worse and hard to treat. In our study, these patients visited our hospital within zero to thirty days after disease onset, with average 8.3 days before the visit. A definitive diagnosis can be made 0–2 days after the visit. Once the diagnosis was made, the delivery was performed on the same day or next day. With increasing emphasis on health, most of patients were admitted in time due to abdominal discomfort, resulting in mild conditions or no serious complications. After a combined treatment including early termination of pregnancy, protection of liver and kidney function, blood transfusion, anti-infection and supportive care, overall patients have a short hospital stay and an improved prognosis. However, serious complications-abdominal hemorrhage and hemorrhagic shock caused by DIC occurred in two pregnant women due to a long time between the visit time and the onset time (10 days or 20 days). One patient was successfully rescued with great difficulty, while the other died after unsuccessful rescue. These cases further emphasize the importance of early visit, early diagnosis and early cesarean section.Until recently, the etiology of AFLP has not been fully elucidated. This is partly due to the lack of knowledge about the disease and extremely low incidence of AFLP. Hence, few studies on this aspect have been done. Based on current researches, impaired beta oxidation of fatty acids in mother or fetus, particularly defects in the enzyme long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), which breaks down long-chain fatty acids in the liver, have been suggested to link to the development of maternal AFLP –17. IbdaIn summary, early visit, early detection, early termination of pregnancy and multidisciplinary comprehensive treatment are the key factors to improve the prognosis of AFLP patients. Contrary to common genetic mutant sites in Western people with AFLP, to our knowledge, although limited size of study, this report is the first to present the lack of common mutation involved in fatty acid oxidation in Chinese patients with AFLP via whole-exome sequencing. However, further studies are needed with larger and more varied samples to validate the conclusion.Additional file 1. General characteristics of the 13 cases of AFLP.Additional file 2. Laboratory findings and imageological examinations of AFLP patients.Additional file 3. The mutation sites filtered and annotated using GATK variant filtration and ANNOVAR in five AFLP patients.


Recent advances in molecular biology have been successfully applied to the exploration of microbiota from various fluids. However, the urinary microbiota remains poorly explored, as its analysis requires specific technical considerations. Indeed, urine is a low microbial biomass environment, in which the representativity of each bacterium must be respected to obtain accurate data. Thus, sensitive extraction methods must be used to obtain good quality DNA while preserving the proportions between species. To address this, we compared the efficiency of five extraction methods on artificial urine samples spiked with low amounts of four bacteria species. The quality of the DNA obtained was further evaluated by different molecular biology approaches, including quantitative PCR and amplicon-based next-generation sequencing (NGS). Although two extraction methods allowed DNA of sufficient quality for NGS analysis to be obtained, one kit extracted a larger amount of DNA, which is more suitable for the detection of low-abundant bacteria. Results from the subsequent assessment of this kit on 29 human clinical samples correlated well with results obtained using conventional bacterial urine culture. We hope that our work will make investigators aware of the importance of challenging and adapting their practice in terms of the molecular biology approaches used for the exploration of microbiota. Until the recent discovery of a unique urinary microbiota in the urinary tract ,2,3, uriStandard urine cultures have long been used in clinical microbiology laboratories to detect uropathogens, especially during a urinary tract infection (UTI) episode. However, these analyses, which allow the detection of viable bacteria, also exhibit several important limitations: they are less adapted to the detection of anaerobic and slowly growing microorganisms and do not allow the detection of unknown pathogens. Although there have been recent improvements to urine culture techniques, particularly modification of culture media and identification of isolated species by matrix-assisted laser desorption ionization–time of flight mass spectrometry , these sHowever, for molecular tools, and especially sequencing techniques, DNA extraction is a critical step for obtaining accurate data. Indeed, in the case of the extensively studied gut microbiota, the extraction technique employed to extract DNA has been shown to impact results ,15. A laLactobacillus delbrueckii and Enterococcus faecalis were spiked at a high abundance, Prevotella bivia and Escherichia coli at a low abundance. Two separate pools, Pool 1 and Pool 2, were generated and then diluted at 1:10 or 1:20, respectively, to evaluate the performance of the kits when there were different amounts of bacteria in the sample. Equal volumes of the four established pools were subjected to parallel extraction with five commercial methods  experiments using primers specific to each spiked bacterium. Whatever the bacteria and their abundance in the mixture, the BT kit resulted in the lowest cycle threshold (Ct) value, meaning that it recovered the highest amount of DNA . For theE. coli C and by P. bivia D. The amP. bivia .Overall, our results indicate that the BT kit seems able to recover more DNA than the other kits tested, regardless of the bacteria and their representativity in a mixture.p < 1 × 10−4, Pearson’s correlation test). Next, we evaluated whether the quality of DNA extracted could influence taxonomic assignment. Interestingly, a large proportion of reads generated from DNA extracted from the most concentrated extract (Pool 2) with the BI kit, and from DNA extracted from both Pool 1 and Pool 2 with the BT kit, could be attributed to an operational taxonomic unit (OTU)  with a quality score >Q30. Since the pool samples diluted at 1:10 or 1:20 were not concentrated enough to be analyzed using this NGS approach, only non-diluted pool samples were used (data not shown). Bioinformatics analysis of the NGS experiments revealed a significant correlation between the number of filtered reads and the amount of DNA extracted (it (OTU) B. For thit (OTU) . Of partit (OTU) B.We then analyzed the different taxa present in and their representativity within the pools . The fouE. coli were collected and DNA was extracted with the BT kit. The level of E. coli DNA in the samples was firstly measured by qPCR. No analyzable signal (>37 Ct) was detected for the 16 HP samples, whereas a clear signal was obtained for the 13 UTI samples, with Ct values ranging from 7.85 to 34.82 , underscoring the accuracy of the BT kit for DNA extraction from urine samples.We further evaluated the ability of the BT kit to detect the different types of bacteria present in complex human specimens by use of human clinical urine samples. Their infectious status had been previously determined using conventional bacterial urine culture. For this purpose, 8 mL of urine samples obtained from 16 healthy patients (HP) and 13 patients with UTIs caused by to 34.82 . InteresEnterobacteriaceae, with a high proportion of attributed reads. Moreover, the number of assigned reads per sample significantly correlated with the CFU measured by conventional bacterial urine culture . In addition, the families detected differed between the two group of samples. The Enterococcaceae and Streptococcaceae families were the most prevalent  and abundant families in HP samples, while they were scarce in UTI samples . Conversely, the Lactobacillaceae family was highly prevalent in UTI samples (7/8) but was almost completely absent in HP samples (1/5). Moreover, when we measured the microbial diversity in the 13 samples analyzed, we found no significant difference in the Shannon diversity index between the HP and UTI samples, although this is likely to be because of the low number of samples analyzed per group, as a tendency towards significance was observed  urine is composed of a very low microbial biomass compared with other types of samples; (ii) samples have different salt concentrations that may disturb DNA extraction ; and  obtained from clinical samples and E. faecalis (Gram-positive) obtained from ATCC 29212 at a high abundance; P. bivia, (Gram-negative) obtained from clinical samples and E. coli (Gram-negative) obtained from ATCC 25922 at a low abundance. The first extract  was used pure or diluted at 1:10 and the second  pure or diluted at 1:20.Urine from a healthy male volunteer was sterilized by ultrafiltration with a 0.2 µm asymmetric polyethersulfone membrane filter . The absence of bacteria was tested by plating 200 µL both on Columbia agar with 5% sheep blood and Chocolate agar . These were incubated at 35 °C for 4 days under aerobic and anaerobic standard conditions. The sterilized urine was then separated into 2 extracts, which were spiked with 4 bacteria at different abundances: g for 10 min at 4 °C, and the pellet was subjected to DNA extraction using five commercial methods. Each condition was performed in triplicate. The methods used were: (1) QIAmp BiOstic Bacteremia kit ; (2) QIAmp DNA Microbiome kit ; (3) AMPure XP ; (4) DNeasy Blood and Tissue kit ; (5) MagNA Pure Compact Kit  (1 mL of each extract was centrifuged at 5000×  France) . ManufacMidstream urine samples from healthy patients or patients with UTI were collected. DNA from these samples were extracted with the Qiagen DNeasy Blood and Tissue kit (BT kit).The resulting extracted DNA, from both artificial and clinical samples, underwent qPCR using the SYBR Green I Master (Roche) in combination with a LightCycler 480 Instrument (Roche). Specific primers were validated on pure boiled bacteria . ResultsThe samples underwent a PCR amplifying the V1–V3, V3–V4, V4–V5, and V6–V8 regions of the 16S rRNA gene . RegionsRaw sequences were processed and filtered with DADA2 (v1.16) on Rstudio . Brieflyp-values were less than 0.05.Statistical analysis was carried out using Rstudio. Spearman’s rank correlation test, Pearson’s correlation test, and the Mann–Whitney U test were considered to be statistically significant if


There is mounting evidence for the therapeutic use of faecal microbiota transplant (FMT) in numerous chronic inflammatory diseases. Germ free mice are not always accessible for FMT research and hence alternative approaches using antibiotic depletion prior to FMT in animal studies are often used. Hence, there is a need for standardising gut microbiota depletion and FMT methodologies in animal studies. The aim of this study was to refine gut decontamination protocols prior to FMT engraftment and determine efficiency and stability of FMT engraftment over time.C57BL/6J mice received an antibiotic cocktail consisting of ampicillin, vancomycin, neomycin, and metronidazole in drinking water for 21 days ad libitum. After antibiotic treatment, animals received either FMT or saline by weekly oral gavage for 3 weeks , and followed up for a further 5 weeks. At multiple timepoints throughout the model, stool samples were collected and subjected to bacterial culture, qPCR of bacterial DNA, and fluorescent in-situ hybridisation (FISH) to determine bacterial presence and load. Additionally, 16S rRNA sequencing of stool was used to confirm gut decontamination and subsequent FMT engraftment.Male Antibiotic treatment for 7 days was most effective in gut decontamination, as evidenced by absence of bacteria observed in culture, and reduced bacterial concentration, as determined by FISH as well as qPCR. Continued antibiotic administration had no further efficacy on gut decontamination from days 7 to 21. Following gut decontamination, 3 weekly doses of FMT was sufficient for the successful engraftment of donor microbiota in animals. The recolonised animal gut microbiota was similar in composition to the donor sample, and significantly different from the Sham controls as assessed by 16S rRNA sequencing. Importantly, this similarity in composition to the donor sample persisted for 5 weeks following the final FMT dose.Our results showed that 7 days of broad-spectrum antibiotics in drinking water followed by 3 weekly doses of FMT provides a simple, reliable, and cost-effective methodology for FMT in animal research. The human gut harbours trillions of microbes which include bacteria, archaea, viruses, and eukaryotes. Bacteria and archaea make up most of the gut microbiome, and studies have demonstrated a role for the gut microbiota in influencing disease processes in many organs , 2. UnsuAnimal models are increasingly used to study the effect of gut microbiome manipulation by FMT on disease pathogenesis, and gut based therapeutic interventions. Although microbial similarity between mice and humans at the phylum level is remarkable, at the genus level 85% of murine sequences represent species that have not been detected in humans . “HumaniFor animal-based FMT studies, recipient animals are required to be either germ free (GF) or depleted of gut bacteria using antibiotics. Germ free mice are considered the gold standard as they are completely devoid of microbes and are therefore perfect candidates for receiving FMT. However, GF animals display an immature and underdeveloped lymphoid system compared with animals living in a conventional microbiological environment . In addiThe antibiotic-based approach is usually preferred, as it avoids several issues seen in GF models, allowing mice to undergo normal colonisation and full postnatal development of their immune system , 18. HowSimilarly, in animal studies, the frequency and duration of FMT administration have varied from a single administration , to onceIn this study we aimed to address these gaps in the literature, by developing a gut decontamination protocol and determining the minimum duration of antibiotic treatment required to efficiently deplete mice of their commensal gut microbiota. This then enables us to determine the effectiveness of FMT and engraftment stability of human donor microbiota until the end of our follow up period.C57BL/6J mice were obtained from Australian Biological Resources  and allowed to acclimatise for 7 days. Mice were co-housed in a temperature-controlled facility (22–24°C), 12:12 hrs light/dark cycle, with standard irradiated chow  and water available ad libitum. All mice (n = 20) received antibiotic treatment for 21 days. Day 0 corresponded to pre-antibiotics phase. After antibiotic treatment mice were randomised to faecal microbiota transplant (FMT) or Sham groups (n = 10 mice per group)  . AnimalsAnimal experiments were approved by the Animal Care and Ethics Committee  and conducted in compliance with the NSW Animal Research Act 1985 and the National Health and Medical Research Council .ad libitum. This combination of antibiotics is the most commonly used cocktail in literature  of faecal solution (FMT group) or an equivalent volume of Saline (Sham group) was administered to mice via oral gavage once a week for 3 weeks.Faecal microbiota solution was prepared from fresh human stool sample. Stool was collected and homogenised in phosphate buffered saline (PBS) (0.1 g/mL) within an hour of collection. The mixture was passed through a 100 μm cell strainer, followed by a 40 μm cell strainer, and was stored in 15% glycerol/PBS at −80in-situ hybridisation (FISH), qPCR and 16S rRNA sequencing. Stool samples were collected before starting antibiotics on day 0 and every 3–4 days during antibiotic treatment . Stool samples were also collected before inoculation of faecal microbiota (F0) and after each of the three doses of FMT which were administrated once a week for 3 weeks . Post FMT completion, to determine efficiency and stability of gut engraftment over time, stool samples continued to be analysed weekly, for a further 5 weeks, corresponding to the engraftment time points (E)1-E5  were collected aseptically for bacterial culture, fluorescence  (E)1-E5 . Stool pg for 2 min and the supernatant resuspended in 15% glycerol. A 1:1000 dilution of the stool homogenate was prepared and streaked onto blood agar plates [Columbia Agar Base  + horse blood (10% vol/vol)] and incubated at 37°C under aerobic, anaerobic and microaerophilic conditions for 48 h before being observed for growth.To assess bacterial levels in stool pellets, freshly collected pellets were homogenised with 20x volume of PBS, using Qiagen Tissuelyzer II (30 Hz for 1 min). The homogenate was then centrifuged at 300 x 2O, air dried, and mounted with soft mounting media.A universal 16S rRNA probe for bacteria was used for hybridisation (EUB338). Stool pellets were initially homogenised with 20x volume of PBS. FISH was performed as described previously , 27. In DNA from stool samples was extracted using the PSP Spin Stool DNA plus kit  according to the manufacturer's instructions. Total genomic DNA concentration was determined fluorometrically using Qubit dsDNA broad range assay kit .Assessment of bacterial load remaining following antibiotic treatment was performed by qPCR. This was performed using PowerUp SYBR Green Master Mix  with universal 16S primers 926F and 1062R  on the QuantStudio 7 Flex  with the following cycling conditions: initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, with a ramp rate of 1°C/s. This was followed by a melt curve analysis from 65 to 95°C at a rate of 0.5°C/s. Each sample was analysed in duplicate. A positive and negative control were run in each plate. Based on the amplification curve, Ct values for each sample were calculated by the instrument when Ct values were below 40.Library preparation and sequencing was performed using 341F and 805R primers  for the 16S V3–V4 rRNA region on the Illumina MiSeq platform with paired 300 bp reads at Ramaciotti Centre for Genomics .16S rRNA gene forward and reverse reads were imported into Qiime2 . The DADpost-hoc Tukey's multiple comparisons test. P values of < 0.05 were considered statistically significant. Statistical analyses were performed with Prism v8.4 .Results are presented as mean ± SD. Statistical analyses for DNA quantification and qPCR were assessed by one-way ANOVA and post-hoc test was used to identity individual variations in alpha diversity between treatment groups. Distance based permutation multivariate analysis of variance (PERMANOVA)  corrected with 0.05 as the cut off. Corrected P values are presented as q-values.A Kruskal–Wallis test with Dunn's RMANOVA)  was perfp < 0.001)  . At day < 0.001) . No obse< 0.001) , water i< 0.001)  or stool< 0.001)  when comNext, we determined the optimal antibiotic treatment period for gut decontamination. To evaluate the efficacy of broad-spectrum antibiotic on bacterial depletion, bacterial culture, qPCR and FISH of stool aliquots were undertaken to measure bacterial load. Prior to antibiotic treatment, at day 0 (baseline), measures of high bacterial presence were observed across all assays as expected. Numerous colony forming units (CFUs) were observed on blood agar plates , DNA conp < 0.00001; p < 0.0001; At day 3 of antibiotic treatment, no CFUs were visible on aerobic and microaerophilic plates, while a dramatic reduction in CFUs was observed on anaerobic plates . Total sFrom day 7 of antibiotic treatment, there were further reductions in bacterial presence, which remained constant until day 21. At all 5 timepoints between day 7 and day 21 (inclusive), no bacterial growth was observed , total DFollowing antibiotic gut decontamination, we next investigated the degree of recolonisation by oral administration of either donor faeces (FMT) or saline (Sham) for 3 weeks. There was no impact of FMT or Sham on body weight .q < 0.05; q < 0.05) and was sustained for a further 5 weeks to the end of the study  (q < 0.05), indicating no cross-contamination of the control mice from environmental input  , indicatal input . Taxonomal input , again sGF mice are ideal models for gut microbiota studies. However, GF models harbour immunological defects, and the feasibility of additional housing requirements render it challenging for many studies. Thus, broad-spectrum antibiotic depletion of the gut microbiota in animals is used as an alternative. However, there are inconsistencies in the method, dose, and duration of treatment regimes. In this study, we determined the optimal duration required for effective gut decontamination as well as frequency of FMT required to achieve effective gut engraftment by donor FMT. We tested for the presence of faecal bacteria at multiple timepoints by multiple modalities using bacterial culture, qPCR, and FISH analysis. Our results showed that remnants of bacteria were present after three days, and that the optimal duration of antibiotic treatment for gut decontamination was seven days. We also showed that three weekly doses of FMT were sufficient for successful engraftment following antibiotic gut decontamination, and that following FMT, bacteria from the donor persisted in animals for the entire five weeks of follow up.Indeed, antibiotic depletion of microbiota is necessary for the successful engraftment of FMT  as microWe observed a reduction in bacteria  within three days with qPCR assessment confirming optimum bacterial DNA depletion was achieved following seven days of antibiotic therapy. This was in concordance with other studies that administered antibiotics via oral gavage or drinking water and observed depletion of the gut microbiota within four days. In this setting, extending the antibiotic regimen beyond seven days did not improve the efficiency of bacteria depletion . NotablyWe noted that bacteria that remained following antibiotic treatment were not those that were initially abundant in untreated mice, but rather represented residual taxa that were resistant to antibiotic treatment. This supported previous observations that antibiotic treatment exerted a selective pressure on bacterial communities , leadingAddressing the question of the frequency and intervals of FMT to achieve successful engraftment is also important. A singular FMT inoculation was shown to elicit no clear improvements in microbial diversity when compared to saline alone , 39. TheDespite our results showing good engraftment of donor microbiota, the study does have limitations. A longer follow up period could have determined the length of persistence of successful engraftment in mice. Although there is compelling evidence for the use of antibiotics in successful FMT engraftment, we did not compare our results to mice without antibiotic decontamination, nor to germ free mice due to feasibility.Although our gut depletion protocol is likely applicable across different groups of mice, other factors need to be taken into consideration when interpreting results utilising gut depletion and subsequent FMT. We have only used young, male C57Bl/6J mice, which are the most widely used inbred, general-purpose strain of mouse as they are a stable strain and easy to breed , 41. NevThe aim of this protocol was to deplete gut bacteria, which is arguably independent of the mouse strain, age and sex. One caveat would be the residual gut microbiota post-antibiotics may be different between mice of different strains, ages and sexes which is an inherent limitation of antibiotic-mediated gut depletion protocols. This would arise from differences in the initial colonisation of the gut microbiota prior to antibiotics treatment. These differences may result in different immune and inflammatory responses  and lingWe describe a viable and efficient protocol for gut decontamination and microbiota manipulation studies, consisting of seven days of antibiotic administration in drinking water, followed by three weekly doses of faecal transplant into recipient mice. This approach to decontaminate the gut prior to FMT was cost-effective and less invasive, providing an alternative to using GF mice. Standardising methodologies for FMT in animal research are required for the effective translation of pre-clinical findings.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA765053.The original contributions presented in the study are publicly available. This data can be found here: The animal study was reviewed and approved by Animal Care and Ethics Committee, UNSW Sydney.This study was performed in collaboration among all authors. NA designed the study, drafted the manuscript, and performed the statistical analyses. NA and AR collected the animal data. NA, AR, and SKh performed the experimental work. NA and EM performed the microbiome analyses. NA, GH, and AZ analysed the results and supervised the study. SS and SKo revised the manuscript and made significant changes during the review process. All authors contributed to revision of the final manuscript.This study was supported by Sir Owen Glenn grant, RG182137, UNSW. The grant body had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.