In December 2019, cases of unidentified pneumonia with a history of exposure in the Huanan Seafood Market were reported in Wuhan, Hubei Province. A novel coronavirus, SARS-CoV-2, was identified to be accountable for this disease. Human-to-human transmission is confirmed, and this disease ) spread rapidly around the country and the world. As of 18 February 2020, the number of confirmed cases had reached 75,199 with 2009 fatalities. The COVID-19 resulted in a much lower case-fatality rate (about 2.67%) among the confirmed cases, compared with Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Among the symptom composition of the 45 fatality cases collected from the released official reports, the top four are fever, cough, short of breath, and chest tightness/pain. The major comorbidities of the fatality cases include hypertension, diabetes, coronary heart disease, cerebral infarction, and chronic bronchitis. The source of the virus and the pathogenesis of this disease are still unconfirmed. No specific therapeutic drug has been found. The Chinese Government has initiated a level-1 public health response to prevent the spread of the disease. Meanwhile, it is also crucial to speed up the development of vaccines and drugs for treatment, which will enable us to defeat COVID-19 as soon as possible. In December 2019, a cluster of pneumonia of unknown etiology was detected in Wuhan City, Hubei Province of China. The first 27 reported cases were all related to Huanan Seafood Wholesale Market, which sells aquatic products, live poultries, and wild animals ,4. The COn 31 December 2019, Wuhan Municipal Health Commission reported a number of unknown pneumonia cases related to Huanan Seafood Wholesale Market, 27 cases were hospitalized, seven of which were in serious condition . On Jan Since 16 February 2020, the cumulative COVID-19 case number increased quickly; meanwhile, the daily emerging case number increased steadily to 3886 on 4 February 2020, and then fluctuated to 2015 on 11 February 2020. The fatality cases number increased steadily to 2004 cases on 18 February 2020. The cumulative and daily emerged cases number jumped to 59,804 and 15,152, respectively, on 12 February 2020 . This fiThe COVID-19 resulted in much lower mortality (about 2.67% up-to-date) among the confirmed cases, compared with Severe Acute Respiratory Syndrome (SARS) at 9.60% (November 2002–July 2003) and Middle East Respiratory Syndrome (MERS) at 34.4% (April 2012–November 2019) . The medAccording to the “Diagnosis &Treatment Scheme for Novel Corona Virus Pneumonia  6th Edition”, the source of infection is majorly the COVID-19 patients, even the asymptomatic patients can also be the source of infection. The transmission way is majorly through respiratory droplets and contacting. People are generally susceptible to this virus.Coronavirus is a single strand positive RNA (+ssRNA) virus, belonging to order Nidovirales, family Coronaviridae, and subfamily Orthocoronavirinae . AccordiCoronavirus has an envelope, the particles are round or oval, often pleomorphic, with a diameter of 50–200 nm . S proteThe knowledge of the physical and chemical characteristics of coronaviruses mostly comes from the study of SARS-CoV and MERS-CoV. The coronaviruses are sensitive to heat and can be killed at 56 °C for 30 min. In addition, ether, 75% ethanol, chlorine disinfectant, peracetic acid, and chloroform can effectively inactivate the virus, but not chlorhexidine .According to the “Diagnosis & Treatment Scheme for Novel Coronavirus Pneumonia  6th Edition” enacted by the National Health Commission of the People’s Republic of China on 19 February 2020, the incubation time after exposure is about 1–14 days [In the early stages of the disease, the total number of leukocytes in peripheral blood is normal or decreased, the lymphocyte count is decreased, and some patients present elevated levels of liver enzyme, muscle enzyme, and myoglobin; some severe cases present elevated troponin level. Most patients show elevated C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and normal procalcitonin. In severe cases, the patients present with increased D-dimer and progressively decreased peripheral blood lymphocyte . CompareAll patients suffered from pneumonia, and chest CT scans showed shadows in the lung . In the To provide more information about the disease for the treatment in clinics, we collected detailed information about the fatal cases released by official channels. A total of 41 fatal cases were used to reveal the symptoms of the deaths, showing the symptom composition of fever (80.5%), cough (56.1%), short of breath (31.7%), chest tightness/pain (24.4%), fatigue (22.0%), dyspnea (12.2%), dizziness/headache (4.9%), general pain (7.3%) and chills (4.9%) . A totalThe diagnosis was based on a set of clinical criteria recommended by the National Health Commission of the People’s Republic of China and the National Administration of Traditional Chinese Medicine .The cases comply with any item of A and any two items of B, or with 3 items of B as follows. (A) Epidemiological history: within two weeks before disease onset, 1. have a history of travel or residence in the district with case report; 2. have contacted the patients positive with nucleic acid detection; 3. have contacted with the patients with fever and respiratory symptoms from the district with case report; 4. disease onset in clustering. (B) Clinical manifestations: 1. Have fever and/or respiratory tract symptoms; 2. Have pneumonia with image characteristics mentioned above; 3. In early stage of the disease, have normal or decreased total number of leukocytes, or decreased lymphocyte count.The unconfirmed cases met the criteria of the suspected cases and are identified positive with SARS-CoV-2 RNA, by real-time RT-PCR or gene sequencing, from the sputum, throat swab, lower respiratory tract secretion, or other samples collected from patients.Mild cases: have mild symptoms, no pneumonia manifestation in chest image;Common cases: have fever, respiratory symptoms, and pneumonia manifestation in chest image;2: arterial partial pressure of oxygen, FiO2: fractional concentration of inspired oxygen);Severe cases: comply with any item of the follows. (A) dyspnea, respiratory rate ≥30 times/min; (B) at resting state, finger oxygen saturation ≤ 93%; (C) PaO2/FiO2 ≤ 300 mmHg  show respiratory failure and mechanical ventilation is required; (B) present with shock; (C) combine with other organ failure needing Intensive Care Unit (ICU) monitoring and treatment; (D) chest imaging shows multilobe lesions or progress of lesion focus within 48 h ≥ 50%; (E) combine with other clinical conditions requiring hospitalization.Suspected and confirmed cases should be treated in isolation in hospitals with effective isolation and protective conditions. The suspected cases should be isolated in a single room, and the confirmed cases can be accepted in the same room. Critical cases should be treated in ICU as soon as possible.Bed rest, strengthen supportive treatment, ensure sufficient energy; pay attention to water-electrolytes balance and maintain the stability of the internal environment; closely monitor vital signs and finger oxygen saturation, and so on.Monitor the blood routine, urine routine, C-reactive protein (CRP) and health indications , coagulation function, arterial blood gas analysis if necessary, and recheck chest imaging.According to the change of oxygen saturation, give effective oxygen therapy in time, including oxygen given by nasal catheter or mask. If necessary, apply high flow oxygen therapy via the nose, noninvasive or invasive mechanical ventilation, and so on.Antiviral treatment: no effective antiviral drug at present. Treat with IFN-α aerosol inhalation , and/or Lopinavir/Ritonavir oral administration .Antibiotic treatment: avoid blind and improper use of antibiotics, especially the combination use of broad-spectrum antibiotics. Strengthen bacteriological monitoring. Antibiotics should be used in time in secondary bacterial infection.Treatment principle: based on symptomatic treatment, actively prevent and treat complications, treat basic diseases, prevent secondary infection, and timely apply organ function support.Respiratory support: apply noninvasive mechanical ventilation for two hours, if the condition is not improved, or the patient is intolerable to noninvasive ventilation, accompanied with increased airway secretions, severe coughing, or unstable hemodynamics, the patient should be transferred to invasive mechanical ventilation in time. The “lung-protective ventilation strategy” with low tidal volume should be adopted in invasive mechanical ventilation to reduce ventilator-associated lung injury. If necessary, ventilation in the prone position, recruitment maneuver, or extracorporeal membrane oxygenation (ECMO) can be used.Circulation support: improve microcirculation based on full fluid resuscitation, use vasoactive drugs, and apply hemodynamic monitoring if necessary.Others: according to the degree of dyspnea and the progress of chest imaging, use glucocorticoids appropriately for a short time (3–5 days) with the recommended dose no more than what is equivalent to methylprednisolone 1–2 mg/kg·day.As of 26 January 2020, 30 provinces have initiated a level-1 public health response to control COVID-19 . A levelThe responsibilities of the organizations are shown in The recent outbreak of the unknown severe pneumonia in China is caused by a novel coronavirus named 2019-nCoV [In 2002 and 2003, the outbreak of SARS brought a disaster to the people of the world, especially the Chinese people ,25. FortCOVID-19 appeared just one month before the Spring Festival of China, and the massive population flow has brought great challenges for disease prevention and control. This virus can be transmitted from human to human and no effective treatment drug has been found. The most effective prevention and control measures are to find suspected patients and close contacts, confirm patients and virus carriers, and block the transmission through isolation, disinfection, and personal protection. Therefore, early detection, isolation, and treatment of patients are the key measures to control the source of infection and reduce the infection rate. It is also crucial to avoid nosocomial infection by strengthening the management of medical staff and patients. Health education on knowledge for disease prevention and control is also important. Finally, if we want to eliminate the threat of this novel coronavirus pneumonia similar to SARS, we need to learn more about the pathogenesis of the virus and develop specific vaccines and therapeutic drugs as soon as possible.


Chara vulgaris spermatids differentiation. The effect of a bromodomain inhibitor, JQ1 (100 μM), on the distribution of individual stages of spermatids and their ultrastructure was studied. The material was Feulgen stained and analysed in an electron microscope. JQ1 caused shortening of the early stages of spermiogenesis and a reverse reaction at the later stages. Additionally, in the same antheridium, spermatids at distant developmental stages were present. On the ultrastructural level, chromatin fibril system disorders and significantly distended endoplasmic reticulum (ER) cisternae already at the early stages were observed. Many autolytic vacuoles were also visible. The ultrastructural disturbances intensified after prolonged treatment with JQ1. The obtained data show that JQ1 treatment led to changes in the spermatid number and disturbances in chromatin condensation and to cytoplasm reduction. The current studies show some similarities between C. vulgaris and mammals spermiogenesis. Taken together, these results suggest that JQ1 interferes with the spermatid differentiation on many interdependent levels and seems to induce ER stress, which leads to spermatid degeneration. Studies on the role of bromodomains in algae spermiogenesis have not been conducted so far.Bromodomain containing (BRD) proteins play an essential role in many cellular processes. The aim of this study was to estimate activity of bromodomains during alga  Chara vulgaris, this process is the second, following the proliferative phase, stage of spermatogenesis. It lasts 7 days during which 10 stages (I–X) of different duration times are distinguished. During each of the stages, there are characteristic ultrastructural changes [Spermiogenesis is one of the most complex and highly specialized morphogenetic processes. In alga  changes ,3.Drosophila, human and mouse). These proteins have two bromodomains. In mammals in BET proteins, four members of bromodomain containing (BRD) proteins: BRD2, BRD3, BRD4, and BRDT  were distinguished [Arabidopsis, in which 29 BRD proteins have been identified [Brachypodium [Bromodomains, 110-amino-acid domains, are present in many chromatin-associated proteins, histone acetyltransferase and subunits of ATP-dependent chromatin remodeling complexes ,5. Reseanguished ,8. Analynguished . Plants hypodium , tomato hypodium  as well hypodium  hypotheshypodium .Cyanidioschyzon merolae, Porphyra and Chondrus), Chlorophyta (Chlamydomonas reinhardtii) and recently in Charophyta (Chara vulgaris) [Cyanidioschyzon merolae (nine proteins) [Micromonas pusilla  [C. vulgaris (Brg1) [C. reinhardtii where the protein is characterized by the presence of as many as three bromodomains [Thalassiosira pseudonana (strain CCMP1335) bromodomains in 27 predicted proteins were identified [BRD proteins play an important role, among others, in controlling leaf development , in the ulgaris) . There iroteins) , Micromoroteins) , C. vulgs (Brg1) , and in odomains ; in addientified .Brdt gene caused sterility of these mammals [Brdt gene in two fish species, which have different methods of nuclear protein exchange, revealed variations in this gene expression which could indicate a different role of Brdt protein [Dicentrarchus labrax) follows the same scheme as in Chara [C. vulgaris is a model organism which was earlier applied in the study i.e., on different processes during spermatogenesis [Inhibitors for the BET family of bromodomains have therapeutic potential and researches are conducted on many animal cancer cell models  . Among t mammals ,25. In i mammals . Bromodo mammals . The res mammals ,28. The  protein . The excin Chara . Therefoogenesis . This alogenesis ,32.The aim of the current work was to find out what role bromodomains play in the course of spermatid differentiation in this alga. The present research focused on whether and to what extent blocking the activity of bromodomains under the influence of their inhibitor, JQ1, affects the distribution of individual stages of spermatids and the ultrastructure of spermatids during spermiogenesis. To the best of my knowledge, this paper presents the first research on bromodomains in algae spermiogenesis.Chara vulgaris from III–V node pleuridia . The algae were grown in an artificial pond located in the Rogów Arboretum (Poland). Prior to the studies, the algae were grown for a few days in tanks containing water from the natural environment at room temperature under natural light.The research material was antheridia of C. vulgaris, antheridial filaments were developing inside the multicellular antheridium, the concentration of the inhibitor used was many times higher in comparison with the cell line model. Initially the concentration of 50 μM was used, however, barely noticeable changes appeared only in single spermatids.The thallus fragments carrying antheridia were treated for 24 h and 48 h with a bromodomain inhibitor, (+)-JQ1 (hereafter referred to as JQ1)  at 100 μM concentration. The solution of the inhibitor was prepared with the water from the natural environment, with DMSO (Sigma) in the ratio 20 mg/mL. These duration times of treatment with the bromodomain inhibitor were used, because previous analyses ,28 were v/v) for 1 h, rinsed in 96% ethyl alcohol, and kept in 70% ethyl alcohol. Next, the fixed specimens were stained with the Schiff’s reagent (Feulgen staining) according to the standard method . Next, 4 in the same buffer for 2 h. After dehydration in an alcohol series, the material was embedded in Epon 812 and Spurr mixture medium (Polysciences) according to the standard procedure presented earlier [Twenty-four hour and 48 h JQ1-treated material and the control were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) supplemented with 0.007 M CaCl2 for 3 h. The isolated antheridia were gently squashed in a drop of heated 2% agar in cacodylate buffer and postfixed in 1% OsO earlier . Ultrath earlier . All ultAnalyses concerned the transitory stage between proliferation phase and spermiogenesis (64/sp) and spermiogenesis stages (I–X) both in the control and JQ1, a bromodomain inhibitor, treated material. Following 48 h treatment with JQ1, thallus differed in appearance from the control one; however, after acid hydrolysis and Feulgen staining, too few antheridia were obtained from this material, therefore, quantitative analysis of spermiogenesis stages was not possible. The control frequency of spermatids at the stages I–III is greater than in V–VIII ones, and it is connected with a different duration of spermiogenesis stages . Early sOn the other hand, an increase in the number of spermatids in subsequent stages of spermiogenesis V–VIII was observed. At stage IV and IX, slight decreases in the number of spermatids in the material treated with JQ1, with a slight increase of X at the last stage, were revealed . Light mIn antheridial filaments in the control material A and in Ultrastructural changes in both nucleus and cytoplasm area were observed in most spermatids treated with JQ1 (24 h and 48 h) at a given stage in comparison to the control. In some spermatids, these disorders did not affect the whole cell, that is they were not observed in the nucleus but only in the cytoplasm.TEM image analysis revealed in the treated material only the sporadic occurrence of the fusion of two spermatid nuclei at stage II C with viIn nuclei of certain spermatids at stages II–V, in both time variants of JQ1 treatment, condensed chromatin adhered to the nuclear envelope; however, non-condensed chromatin showed a looser configuration and in its area bright or empty spaces were visible C,D, but In the spermatids after treatment with JQ1 (24 h) already from stage II, both broader endoplasmic reticulum (ER) cisternae and thicker nuclear envelope B,C in coAt stage IV, spermatid nuclei shifted to one of the side cell walls and the condensed chromatin adhered to the nuclear envelope. The same image was characteristic of a very early transition stage between IV and V, at which ER cisternae appeared E. Among Analysis of micrographs revealed in the same antheridial filament sporadic side-by-side occurrence of spermatids at two different stages of spermiogenesis (VI and VIII) C.At stage VI (JQ1 24 h), the whole area of spermatid nucleus was uniform and filled with short chromatin fibrils arranged in different directions, between which there were lighter areas C. It wasAt stage VIII and the last spermiogenesis stages , instead of parallelly arranged long chromatin fibrils A, condenThese changes became more pronounced after 48-h treatment with JQ1  and are Similarly as before, the disturbed cytoplasm was visible. A lot of bright spaces and numerous autolytic vacuoles were found  that werAt all stages after 48 h treatment with JQ1, lack of cohesion of cellular structures of spermatids could be seen; it was difficult to notice cell walls in the antheridial filament cells and sometimes to recognize the typical structure of these spermatids C. HoweveChara is the object of research that has been conducted for many years also in our department [Spermatogenesis in the green algae partment ,19. Prevpartment ,19,28,30BRD proteins take part in many cellular processes. Studies concerning the contribution of bromodomains and their inhibitors to chromatin epigenetic modifications include not only sperm cells but also different cancer cell lines ,37,38,39C. vulgaris, antheridial filaments containing spermatids, which are developing inside a complicated cell complex, antheridium, are used as the research model; however, in animal studies, mainly cell lines are used. The correct differentiation of spermatids in C. vulgaris is a complex process that requires the interaction of many elements and participation of the chromatin remodeling factor. In this alga Brg1 protein, a functional homologue of Swi2/Snf2 catalytic subunit of the SWI/SNF chromatin remodeling complex was shown [During spermiogenesis, spermatids undergo the transformation from somatic cells into generative ones. In as shown . Westernas shown , and simas shown ,41.Bromodomains participate in the spermatogenesis control system . StudiesIn human cutaneous T-cell lymphoma cell lines, this inhibitor inhibits proliferation, retaining cells in the G0/G1 phase and reducing their number in the S phase. The impact of the inhibitor is stronger in interaction with the histone deacetylase inhibitor SAHA . A similChara, the cell cycle lacks G1 phase (type S + G2 + M). Based on the above information, it can be assumed that, as in cell lines, also in the case of this alga, JQ1 may inhibit proliferation and the reduction of S phase leads to decreased spermatid number at stages 64/sp–III. In addition, after treatment with JQ1 in the same antheridium, asynchronous spermatid differentiation was observed, which might be the result of changes in the earlier proliferative phase. Because previous studies revealed at stages 64/sp–III the most intensive positive immunoreaction with the anti-Brg1 antibody [In the case of the examined algae, the effect of JQ1 on the proliferative phase of spermatogenesis was not analysed. However, a significant percentage decrease in spermatid count at stage 64/sp and at the early stages of spermiogenesis I–III, which is similar to mouse round spermatids, was observed. In antibody , it seemantibody .Chara has a different type of meiosis , which occurs only after fertilization. During spermiogenesis, after treatment with JQ1, a decrease in the number of sperm cells was accompanied by changes of their motility. As a result of a long-term daily treatment of mouse with JQ1, a reduced volume of testis and of seminiferous tubules area was observed. Disturbances during the developmental stages of male reproduction cells in mice did not concern all cells. These changes were visible on histochemical sections of testis tubules and finally led to the formation of incorrect round spermatids, as well as multinucleated symplasts [The studies conducted on male reproductive cells of mammals (man and mouse) showed that bromodomain inhibitor JQ1 blocked BRDT, and the effects of its activity depended on the time of application and its dose. JQ1 affected the developmental stages of these cells already during meiosis and successive post-meiotic stages ,50. In tymplasts .C. vulgaris spermatids after JQ1 treatment, ultrastructural changes concerning the whole cell area were not present in all cells. It is interesting that the fusion of two spermatid nuclei at stage II was also observed. Studies on root meristem cells of Narcissus revealed that this fusion might occur as a result of the incomplete primary septum formation, separating these two cells, in telophase [In elophase . It can elophase , this imC. vulgaris, DNA double-stranded breaks occur at the middle stages of spermatid differentiation and are necessary for the correct rearrangement the chromatin structure from nucleosomal to that connected with protamines. Their lack leads to disturbances in nucleoprotein exchange and chromatin condensation at stages VI–VIII of spermiogenesis [Studies on pancreas tumor cells showed that JQ1 caused DNA damage, the marker of which is γH2AX. The number of fluorescent foci of γH2AX increased in JQ1 in a dose-dependent manner ,47. At Cogenesis .No ultrastructural studies have been conducted so far on spermatids treated with bromodomain inhibitors. On the TEM level, only the impact of a potent bromodomain inhibitor, I-BET-151, on changes in cardiomyocyte mitochondria of male rodents, was shown . This paC. vulgaris spermiogenesis, ER is most developed at the advanced stage V [Chara. In internodal cells of C. vulgaris thallus, the action of heavy metals (mercury) also caused the appearance of dilated ER cisternae [Arabidopsis vacuoles indicating ER stress-dependent autophagy [C. vulgaris. Thus, its dysfunction makes the proper exchange of histones into protamines impossible [The prolonged treatment with JQ1 (48 h) led to the formation of spermatids altered to such an extent that it was sometimes difficult to recognize individual cell structures. Compared to the earlier research ,28, such stage V ,44,54. I stage V . Electro stage V , dilated stage V , and hep stage V  also as isternae . In rootutophagy  and in hutophagy  were obsutophagy , and as utophagy ,63. Probpossible ; howeverThe current studies broaden the knowledge about algae Charophyta spermiogenesis. The results obtained revealed the essential role of bromodomains in this process. The blockage of their proper functioning caused such pronounced disturbances that have not been seen so far after treatment with different inhibitors. These changes both in the nucleus and cytoplasm of spermatid may confirm that JQ1 inhibitor interferes with the spermatid differentiation on many interdependent levels. This issue has not been analyzed in detail on the ultrastructural level until now.


The best drug-like candidates were identified, after parental structure modifications, by their highest affinity to the target protein. We found that our newly designed compound R5, a derivative of the R428 patented analog, is the most promising inhibitor of the Axl kinase according to the three molecular docking algorithms applied in the study. The molecular docking results are in agreement with the molecular dynamics simulations using the MM-PBSA/GBSA implicit solvation models, which confirm the high affinity of R5 toward the protein receptor. Additionally, the selectivity test against other kinases also reveals a high affinity of R5 toward ABL1 and Tyro3 kinases, emphasizing its promising potential for the treatment of malignant tumors.The high level of Axl tyrosine kinase expression in various cancer cell lines makes it an attractive target for the development of anti-cancer drugs. In this study, we carried out several sets of  Receptor tyrosine kinases are transmembrane proteins, which consist of several domains that are activated upon ligand binding to their extracellular regions, triggering downstream signaling cascades  under the reference code 5U6B. Other kinases such as ABL1, ALK5, FYN, JAK, MER, MET, and Tyro3 have been derived under the 4WA9, 3GXL, 2DQ7, 5WO4, 5U6C, 2WD1, and 3QUP PDB codes, respectively. Coordinates for the Axl kinase type I inhibitors have been obtained from the PubChem under the following CIDs: 46215462 (R428), 11282283 (Amuvatinib), 5328940 (Bosutinib), 11626560 (Crizotinib), 49803313 (Gilteritinib), 49870909 (S49076), 46870258 (SGI-7079), 5329102 (Sunitinib), 56839178 (TP-0903), and 73425588 (UNC2025). The PubChem CIDs for the 136 R428 patented analogs as well as the 26 analogs of crizotinib can be found in the All compounds were energy-minimized prior to docking, with the help of the Molecular Operating Environment (MOE) software [Molecular Operating, Environment (MOE), Molecular docking simulations have been performed with the help of well-validated . The AutoDock tools were used to generate the input parameter files for docking. For the current study, the receptor was considered as a rigid molecule, while the ligands contained rotatable bonds. Pure protein was applied for the docking, while all non-protein moieties were removed. Additional hydrogen atoms were added to the receptor, and the new PDB coordinates were saved. The ligand PDB file was modified by the addition of groups representing the Gasteiger charges. The volume of the grid box was set as 50 × 50 × 50 Å, with 0.375-Å spacing. The center of the grid box was placed so that it coincided with the center of the co-crystalized structure of the compound (macrocyclic compound 1). A genetic algorithm was selected to set the search parameters. The number of docking runs was fixed to 50. The conformations with the lowest binding energies have been selected for further analysis.Molecular docking using AutoDock software  and the MOE [Molecular Operating, Environment (MOE), The figures were prepared using the UCSF Chimera  simulations were performed using the AMBER 16 package  have been derived from the above-mentioned designed R6 and R3 compounds with a slight alteration of the template structure. Thus, the compound R7 was obtained by replacing a hydrogen atom with a hydroxyl group in the compound R6. The compound R8, in turn, was obtained by replacing an amino group with a nitroso group in the compound R6. The compound R9 represents a pure template  with D690 see . The comNext, we tried to obtain new compounds with improved binding based on the R428_2, R428_3, and R428_4 structural characteristics. Since these compounds share an almost identical structure and acquire a quite similar conformation inside the pocket see , 3B, we Ki) as well as the ligand efficiencies (LE) for ATP, R428, R428_1, and R5. The data are reported in Ki to be 12 times lower than that of ATP, indicating a highly competitive binding of the R428 over ATP. The compound R428_1 exhibits an even lower Ki than the R428 itself, and, finally, the newly designed compound R5 shows the lowest value of Ki (2.3 nM), which is 19 times lower than that of the R428. The calculated LE for R428, R428_1, and R5 compounds are showing almost the same values; the LE for the ATP is slightly lower than for the above-mentioned compounds. Taken together, these data indicate the high efficiency of the newly designed compound R5.To estimate the strength of our best-designed compound R5, we have compared the calculated inhibition constants (Gbind) based on the implicit solvation models were calculated for R428, its best derivative—compound R428_1, the best-designed compound R5 and ATP as a reference molecule, establishing the best ligand affinity to the Axl kinase. The MM-PBSA/GBSA calculations  or an endothermic (MM-PBSA) binding reaction . NeverthTo establish how selective the best-designed compound R5 toward Axl kinase is, we further performed a selectivity test against a set of other kinases such as ALK5  receptor agonists and later a structure-based drug design strategy for the design of the novel compounds. They came up with six potent baclofen analogs targeted on the activation of the GABAB receptor  to evaluate the binding affinity of the compound R5 to them. Interestingly, this compound has shown the strongest binding properties toward Tyro3, which is another receptor of the TAM family. We tried to explain this phenomenon by performing a comparative analysis for the binding pockets of the high-scoring  and the relatively low-scoring (MET) kinases see . It is cin vitro or in cell culture. Similarly, inhibiting only one cancer pathway may not be the strongest strategy, particularly in view of clinical application. However, there is now consensus that, due to their well-established and clear inhibitory effect on several cancer pathways such as cell survival, invasion, migration, chemo-resistance, and metastasis, such TAM inhibitors hold great promise for cancer treatment (Davra et al., TAM activation can, thus, be efficiently inhibited by the small-molecule inhibitors for this interaction. For the TAM activation blocking compounds RU-301 and RU-302, Kimani et al.  suggesteIt is, of course, possible to get a very high false-positive rate in the virtual screening, but this can mainly happen if you do not do any scoring or accept all targets equally well.In particular, as the reader can see in our manuscript, the chosen strategy strongly reduces the amount of false-positives. We always start from the best possible leads, and that is not only according to the scoring but also, more importantly, according to all available experimental data.Nevertheless, we think that it is necessary to validate the proposed compounds with actual experiments, and that is the reason why we want to publish our results here, considering other purely theory articles such as those of Fu et al. , Duan etin silico selectivity test against other kinases has also shown a high affinity of R5 toward ABL1 and Tyro3. Our in silico results suggest the application of the newly designed compound R5 particularly as an anti-cancer agent. Direct proof by experiment is now the next important step. Further synthesis and in vitro tests of this compound against various cancer cell lines, where Axl, ABL1, and Tyro3 play a significant role in proliferation, division and metastases are further steps to be taken, and further indications for TAM inhibition may follow.In summary, systematic computational studies were performed to establish the best drug candidates for targeting Axl kinase to treat cancer. The first compound selection has shown that the most potent drugs for such a purpose are crizotinib, macrocyclic compound 1, and R428. Further searches were performed based on the analogs of pharmaceutically available drugs, namely, crizotinib and R428. The docking results for crizotinib analogs did not demonstrate scores higher than the original drug itself. Therefore, we focused on crizotinib by modifying it further. The R428 analog docking resulted in four high-scoring compounds, which were further utilized for structural modifications. The structural modifications of crizotinib did not show any significant Axl binding improvements, while modifications of the R428 analogs resulted in a new potential drug candidate—compound R5. Apart from the docking tests, the potency of this newly designed compound as an Axl kinase inhibitor has been confirmed by a molecular dynamics simulation in terms of protein–ligand complex stability. Furthermore, an All datasets generated for this study are included in the article/in silico screening and docking of the compounds, designed the new compounds, performed a selectivity test, analyzed the data, made corresponding figures and tables, and drafted the manuscript. SS performed molecular dynamics simulation, analyzed and reported the corresponding data, made tables, and participated in the manuscript drafting and editing. TD supervised the study, analyzed its data, and participated in the manuscript drafting and editing. All authors finalized the manuscript together.ES planned the study, performed the 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.


Bromhexine hydrochloride tablets may be effective in the treatment of Coronavirus disease 2019 (COVID-19) in children. This study will further evaluate the efficacy and safety of bromhexine hydrochloride tablets in the treatment of COVID-19 in children.The following electronic databases will be searched, with all relevant randomized controlled trials (RCTs) up to August 2020 to be included: PubMed, Embase, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), the Chongqing VIP China Science and Technology Database (VIP), Wanfang, the Technology Periodical Database, and the Chinese Biomedical Literature Database (CBM). As well as the above, Baidu, the International Clinical Trials Registry Platform (ICTRP), Google Scholar, and the Chinese Clinical Trial Registry (ChiCTR) will also be searched to obtain more comprehensive data. Besides, the references of the included literature will also be traced to supplement our search results and to obtain all relevant literature.This systematic review will evaluate the current status of bromhexine hydrochloride in the treatment of COVID-19 in children, to evaluate its efficacy and safety.This study will provide the latest evidence for evaluating the efficacy and safety of bromhexine hydrochloride in the treatment of COVID-19 in children.CRD42020199805.The private information of individuals will not be published. This systematic review will also not involve endangering participant rights. Ethical approval is not available. The results may be published in peer-reviewed journals or disseminated at relevant conferences. Fever, fatigue, and dry cough are the most common clinical symptoms of COVID-19. At present, it is recommended to use a-interferon, lopinavir/ritonavir, ribavirin, chloroquine, and abidol for antiviral therapy.–6 Bromhexine is only used as an adjuvant drug for symptomatic treatment to alleviate the respiratory problems of patients. Literature research and molecular simulation have found that bromhexine may have antiviral potential that has never been discovered before.–9 Combined with the other respiratory effects of bromhexine, it may be used as a first-line drug in the clinical treatment of novel coronavirus.Novel coronavirus (SARS-CoV-2) can be transmitted through droplets, close contact, aerosols, and other forms.,11 Therefore, TMPRSS2 inhibitors can be used to block SARS-CoV-2 infection. As early as 2014, some research teams selected bromhexine as the drug with the strongest inhibitory effect on TMPRSS2. It is suggested that the TMPRSS2 inhibitor bromhexine can be used to block pulmonary virus infection, whereas chloroquine can be used to inhibit the viral lysosome pathway. A large number of sticky secretions have been seen in the autopsy lung sections of COVID-19 patients who have died. Bromhexine can reduce sputum viscosity, promote lung ventilation and expectoration, and remove virus residues.SARS-CoV-2 mainly relies on transmembrane protease, serine 2 (TMPRSS2) to enter the cell.At present, many clinical trials have been carried out to study the efficacy of bromhexine in the treatment of COVID-19 patients. However, the strength of the evidence for the clinical efficacy and safety of bromhexine hydrochloride tablets in the treatment of COVID-19 in children is unclear. Therefore, the purpose of this study is to evaluate the clinical efficacy and safety of bromhexine hydrochloride tablets in the treatment of COVID-19 in children.2 This work has been registered at PROSPERO. The registration number for this study is CRD42020199805.We will conduct this work following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols (PRISMA-P) statement guidelines.2.12.1.1All randomized controlled trials (RCTs) of bromhexine hydrochloride tablet treatment for COVID-19 in children will be included without language restriction. Observational studies, conference abstracts, animal studies, data which are incomplete or contain obvious errors, and letters will all be excluded.2.1.2Inclusion:Aged between 2 and 18 years, male or female;Laboratory (reverse transcription-polymerase chain reaction) and clinical symptom-confirmed cases of COVID-19, according to the official guidelines of the “Pneumonia Diagnosis and Treatment Scheme for Novel Coronavirus Infection ,” or patients with negative 2019-nCoV RNA results but suspected clinical symptoms;The legal guardian of the enrolled children has given informed consent voluntarily .Exclusion:Patients diagnosed with severe or critical pneumonia caused by novel coronavirus infection;Patients allergic to the ingredients of bromhexine hydrochloride tablets and/or with definite contraindications on the label of bromhexine hydrochloride tablets;Patients with a history of serious cardiovascular and cerebrovascular diseases, such as heart failure, congenital heart disease, serious arrhythmia, and cerebrovascular accident;Patients with severe liver disease or liver insufficiencies, such as cirrhosis or chronic active hepatitis;Patients with severe kidney disease ;Patients with a history of malignant tumors;Patients with serious hematopoietic diseases or agranulocytosis.2.1.32.1.3.1Bromhexine hydrochloride tablets + standard treatment.2.1.3.2Standard treatment.2.1.42.1.4.1Rate of aggravation.Clinical remission rate.Rate of patients with fever.Rate of patients with dyspnea.Rate of patients with mechanical ventilation.Rate of ICU admission.2.1.4.2Adverse events.2.22.2.1We will search PubMed, Embase, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), the Chongqing VIP Chinese Science and Technology Database (VIP), Wanfang, the Technology Periodical Database, and the China Biomedical Literature Database (CBM) for all dates up to August 2020. The PubMed search strategy is shown in Table 2.2.2Baidu, the International Clinical Trials Registry Platform (ICTRP), Google Scholar, and the Chinese Clinical Trial Registry (ChiCTR) will also be searched to obtain more comprehensive data. In addition, the references of the included literature will also be traced to supplement our search results and to obtain all the relevant literature.2.32.3.1EndNote X8 will be used for document management. Two researchers will independently sift through the literature, extract data, and cross-check their accuracy. In the aspect of literature screening, the title and abstract of the research retrieved in the retrieval process will first be read, then texts identified as related research will be read in full. Agreement must be reached between the 2 researchers to decide whether to include a text or not, with obviously irrelevant literature immediately excluded. If there is any objection, a third researcher will be consulted to assist in making the judgment. Excel 2019 will be used to create a data extraction table and extract related data. The study selection procedure is shown in Figure 2.3.2The 2 reviewers will independently extract relevant data from eligible studies, and any objections will be resolved through discussion with the third reviewer. The extracted research data will mainly include the first author, year of publication, study location, baseline characteristics of participants, sample size, intervention time, follow-up, outcome measurement indicators, and adverse events. If necessary, the trial author will be contacted for more information.2.4The 2 researchers will independently assess the risk of bias in the inclusion of studies from the Cochrane Library according to the guidelines of the RCTs intervention system evaluation manual. The following items will be considered: random sequence generation, allocation hiding, the blindness of participants and personnel, the blindness of result evaluation, incomplete result data, selective reporting, and other sources of bias. The quality of the study will be classified as “high,” “unclear” or “low” bias risk. If there are any objections, the third researcher will be consulted and a final decision made as required.2.52.5.1The RevMan 5.3 software (provided by the Cochrane Collaboration) will be used for the statistical analysis. For dichotomous variables, relative risk has been selected for use, and all will be represented by effect value and 95% confidence intervals.2.5.2P ≥ .1, it will indicate that there is no inter-study heterogeneity. However, if P < .1, it will indicate that there is inter-study heterogeneity present. At the same time, the I2 statistic will be used to quantitatively evaluate the inter-study heterogeneity. If I2 ≤ 50%, heterogeneity will be considered to be low, and the fixed-effect model will be adopted for use. Conversely, if I2 > 50%, it will indicate that there is significant heterogeneity, in which case the source of the heterogeneity will be explored through a subgroup or sensitivity analysis. If there proves to be no obvious clinical or methodological heterogeneity, then it will be considered as statistical heterogeneity, and the random-effects model will then be used for the analysis instead. Descriptive analysis will be used if there is significant clinical heterogeneity between the 2 groups and subgroup analysis is not available.The Q test will be used to qualitatively determine inter-study heterogeneity, and if 2.5.3Publication bias will be estimated by a funnel plot analysis if sufficient studies are included (>10 studies).2.5.4Subgroup analyses will be carried out based on drug dose and duration.2.5.5To test the stability of the results, we will use the one by one elimination method for sensitivity analysis.2.5.6This systematic review will not require ethical approval because there will be no data used in our study that is linked to individual patients. The results will be disseminated only in peer-reviewed publications.3–19 As a potent inhibitor of TMPRSS2, a key protease in the infection and transmission of novel coronavirus SARS-CoV-2, bromhexine has the advantage of low price and greater safety. Also, bromhexine and its metabolites can competitively bind to cellular receptor angiotensin-converting enzyme 2 (ACE2). This strongly inhibits the key M proteases of novel coronavirus SARS-Cov-2, promotes the release of endogenous active substances in the lungs, maintains alveolar function, and promotes sputum excretion.Bromhexine is a kind of expectorant, which is mainly used in the treatment of chronic bronchitis, emphysema, bronchiectasis, and chronic obstructive pulmonary disease. In addition, several university and institutional experts have jointly published articles to evaluate the importance of TMPRSS2 for respiratory virus infection and introduce the therapeutic potential of bromhexine as a TMPRSS2 inhibitor for COVID-19.Related studies have pointed out that effective drugs to prevent novel coronavirus infection must contain either TMPRSS2 inhibitors or competitive ACE2 binding inhibitors, and it is particularly recommended that bromhexine, a specific TMPRSS2 inhibitor, be used to prevent and treat COVID-19.To our knowledge, this is the first systematic review of the clinical efficacy and safety of bromhexine hydrochloride tablets in the treatment of COVID-19 in children. We believe that this review will provide evidence-based advice for a COVID-19 treatment system in children, optimize clinicians’ treatment strategies, and identify promising treatment options for researchers.Conceptualization: Yuying Wang and Xiaohong RenData collection: Yinghua Zhang and Xia ChenFunding support: Xiaohong RenLiterature retrieval: Kun Xu and Tianjing ZhangSoftware operating: Kun Xu and Tianjing ZhangSupervision: Kun Xu and Tianjing ZhangWriting – original draft: Yuying Wang and Xiaohong RenWriting – review & editing: Yuying Wang and Xiaohong Ren


The objective of this study was to assess the diagnosis value of urinary inflammatory index (UII) and systemic immune-inflammation index (SII) for UTI. Nine inflammatory indexes including neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, SII and six UIIs were calculated for Receiver operating characteristic curve analysis to select which one is suitable for the screening of UTIs or distinguishing the types of bacteria. UII3, which calculated from leucocyte esterase (LE), nitrite, white blood cells and bacteria, was preferentially used as an indicator for the diagnosis of UTI when the threshold was set at 0.53. UII2 was more suitable for the distinction between groups when the cutoff is set to 0.94. Appropriate urinary inflammation index calculated by rapid urinalysis of urine dipstick and urine sediment can help us to predict urinary tract infection and bacterial type, and reduce the workload and costs of urine culture. Escherichia coli is the most important pathogen causing UTI, and other pathogens include Enterococcus, proteus, Klebsiella, Pseudomonas aeruginosa, etc.; hospital acquired UTI pathogens include Staphylococcus, Candida, etc3. Urinary system diseases include asymptomatic bacteriuria, symptomatic UTI and UTI related septicemia. Bacteriuria ≥ 105 CFU/ml is the most basic condition for laboratory diagnosis of UTI4. Other symptoms such as dysuria, increased frequency of urination, hematuria and backache are also strong evidence for diagnosis of UTI5.UTIs are an inflammatory reaction of urinary tract epithelium caused by a wide range of pathogens invasion, including Gram-negative and Gram-positive bacteria as well as fungi, usually accompanied by bacteriuria and pyuria, which affecting about 150 million people worldwide every year6, about 70–80% of urine culture results are negative7. Clinicians often obtain urine cultures in patients without localizing urinary symptoms or positive culture results, will reflect asymptomatic bacteriuria rather than infection, resulting in the overdiagnosis and overtreatment are also well-recognized problems, then de-adoption of routine urine culture testing was called to action8.Quantitative midstream urine culture is the gold standard for the diagnosis of UTI, and antimicrobial susceptibility testing is helpful to guide the clinical treatment. However, although the traditional method of urine culture is time-consuming, due to the lack of other reliable diagnostic indicators or powerful stimulus for antibiotic treatment11. Frequently used screening items such as microscopy analysis for WBCs and bacteria as well as dipstick testing for LE and nitrite in urine are fast but with low sensitivity12. Although the combination of positive test results is very sensitive, the usefulness of the dipstick test alone to rule in infection remains doubtful, even with high pre-test probabilities13. Therefore, rapid screening and accurate prediction of culture results are needed for clinicians shorten the diagnosis time, improve the efficiency of Microbial Laboratory, and win the treatment opportunity for patients as early as possible.To address the shortcomings of urine culture, several researchers are trying to find a rapid screening method that can reduce the necessity of urine culture, which will have a significant impact on the overall turnover time and laboratory economy14, however, they are not widely used in general laboratories. Our study planned to generate a new parameter, UII which was calculated by the rapid test results including urine dipstick and urine sediment analysis, for auxiliary diagnosis of UTI.Although many mature detection methods such as MALDI-TOF mass spectrometry, Fluorescence in situ hybridization (FISH), etc. and emerging diagnostic platforms, such as biosensors, microfluidics, and la-on-a-chip technology, demonstrate the potential for expedited UTI diagnosis using enhanced screening5 CFU/ml bacteria. The average age of male  and female  patients was 71.66 ± 16.60 and 66.17 ± 16.81, respectively (P < 0.05), and the higher infection rate is focused in the age of 50–95  between January 1, 2016, and Dec 31, 2019 produced growth of ≥ 10Escherichia coli is followed in prevalence by Enterococcus, fungi, Klebsiella, Streptococcus, Proteus, Pseudomonas, Staphylococcus, Enterobacter and Acinetobacter enter the top ten, and there was no significant difference in the infection rate among four seasons , there was no significant difference in WBCs between Gram-negative and Gram-positive bacteria (P = 0.6476) . Therefore, we may predict the bacterial type of UTI by UII values and clinical symptoms. Then, we chose UII2, UII3 and UII4 to distinguish the bacterial groups which group A contains Escherichia, Enterococcus, fungi, Klebsiella, Pseudomonas and Enterobacter while group B consists of Streptococcus, Staphylococcus and Acinetobacter. According to ROC curve analysis, AUCs of UII2, UII3 and UII4 were 0.623, 0.647 and 0.642 respectively (P < 0.001). Youden index analysis shows that when we set the cutoff of UII2, UII3 and UII4 at 0.94, 1.81 and 1.52 respectively, the sensitivity and specificity were 81.7% and 62.3% for UII2, 56.1% and 33.6% for UII3, 57.4% and 37.4% for UII4 (P < 0.001). Therefore, UII2 is more suitable for the distinction between groups when the cutoff is set to 0.94 , WBCs and bacteria in urine will increase. In addition to UTI, urinary WBCs may also come from female genital tract pollution, while some patients with low immunity may have normal urinary WBCs, and bacterial may also come from specimen contamination with periurethral, epidermal, perianal, and vaginal flora18 because of the high detection limit of the commercial dipstick. Urinary dipsticks are an effective rapid test for screening for asymptomatic, catheter-associated UTIs in intensive care unit patients11. However, traditional indicators such as LE and nitrite have high specificity but low sensitivity in the diagnosis of UTI, so their limited diagnostic value when used alone does not lead to satisfactory results. In this study, the non-negative rates of nitrite, WBCC, bacteria and ECs were all very low, which was not a powerful evidence for UTI screening, as reported by Koeijers et al.19. Another study reported the nitrite/nitrate ratio determined by the LC-MS/MS was significantly more sensitive (95%) and exhibited a satisfactory specificity (91%) in the screening of UIT20. Although WBCs and LE had high non-negative rates in various bacterial infections, they are not suitable for screening UTI alone. In order to improve the sensitivity and specificity of diagnosis, we combined the indicators related to inflammation in urinalysis to make a comprehensive judgment, so the UII came into being.Rapid urine tests, such as microscopy, for bacteria and WBCs, and dipsticks, for LE and nitrite, are often used to guide early diagnosis and treatment of UTI. Many researchers have tried to compare the correlation between rapid urinalysis and positive urine culture. Generally, LE is more sensitive than the nitrite test in screening for UTI23. However, they seem to have no value in predicting UTIs, this suggests that UTIs are mostly focal inflammation, except for complicated UTI which is often accompanied by systemic diseases such as renal insufficiency, transplantation, diabetes or immune-deficiency4. LE, nitrite, WBCs and bacteria participate in the calculation of UII2, UII3, and UII4. This reminds us that these four indicators play an important role in UTI screening. Unfortunately, the AUC, sensitivity and specificity of each index decreased significantly when we compared to the negative control. Based on our data, only 18.7% (75/477) of patients collected 2 or 3 urine samples for culture, while 4.4% (21/477) of the patients with blood culture or sputum culture were positive, and 59.1% (282/477) of the patients with pyuria. Most of them did not take continuous specimens for testing, or may have been treated with antibiotics before specimens obtaining. Furthermore, a culture that shows significant bacterial growth may not reflect an active infection, for example, asymptomatic bacteriuria occurs in about 10% of community-dwelling older women24. Pyuria is a nonspecific finding that is frequent in older patients with or without bacteriuria25, while the negative predictive value can reach 95% or more to rule out infection if it is absent26.Multiple inflammation indexes such as NLR, PLR, SII and UII were calculated in this study for the screening of UTIs, only UII2, UII3, UII4 and UII6 with a higher AUC (> 0.9) while UII2 had the highest sensitivity of 88.2% and UII4 had the highest specificity of 99.3% at the cutoff of 0.44 and 0.18, respectively. NLR, PLR and SII are widely used in various tumors as useful prognostic indicators27. If we can predict the types of pathogens in advance, it will be of great value for the guidance of early experience drug use. In this study, UII was calculated by rapid urine test for the first time and compared with the results of urine culture. The AUC of 0.927 for prominent applications of UII3 which integrated the characteristics of four items and became the preferred prediction index according to Swets28. To our knowledge, the current study is the only report to focus on the value evaluation of UII in UTI screening.The clinical treatment of UTIs mainly depend on experience and the adjustment of drug use according to the results of antimicrobial susceptibility test. With the spread of antibiotic resistance and its increasing threat to public health caused by UTIs became the second most common diagnosis for empirical antibiotics, national guidelines and antimicrobial management programs have been proposed to meet these challengesUII can be used not only to judge the existence of UTI, but also to roughly identify the types of bacteria. In summary, prediction of UTIs by simple calculation of UII might be helpful in significantly reducing workload and costs of urine culture.5 CFU/ml was considered to be a positive result of a urine culture4. In the instance of culture of > 1 microorganism, only the predominant microorganism was considered19.Urinalysis were performed for urine chemistry and microscopy values by the iRICELL3000 (IRIS diagnostic) automatic urinalysis line which were designed to streamline the UTI testing workflow by generating work lists of possible UTI patient sample values. The indicators measured by iChemVelocity were LE and nitrite, and the iQ200 module assessed were WBCs, white blood cell clot (WBCC), bacteria and epithelial cells (ECs).All patients are required to take midstream urine in sterile containers and submitted for bacterial culture. In the microbial laboratory, the uropathogens were isolated by using 1ul quantitative inoculation ring evenly smeared on the Columbia blood agar plate, cultured at 35 °C for 18–24 h, and continue to culture until 48 h when there is no bacterial growth. Then, they were identified by Vitek-2 compact Complete automated ID/AST platform and MALDI-TOF mass spectrometry , growth of ≥ 1029.Serum concentrations of platelets, neutrophils, and lymphocytes were measured by the UniCel DxH Connected Workcell (Beckman diagnostic). SII, NLR, and PLR were calculated as follows: SII = platelet*neutrophil/lymphocyte, NLR = neutrophil/lymphocyte, PLR = platelet/lymphocyte30. According to the principle of randomization, we selected 500 outpatients or inpatients with negative urine culture results and 500 normal physical examination results as controls, those samples that are tested repeatedly or cultured to be positive again will be removed. Urine and venous blood were obtained and examined in the clinical laboratory Department of Affiliated hospital of Nanjing university of Traditional Chinese Medicine. Data collection was approved by the institutional review board and the Ethics Committee of the First Affiliated Hospital of Nanjing University of Traditional Chinese Medicine and all subjects included in the study gave their informed consent to the scientific use of the data. The methods were carried out in accordance with the relevant guidelines and regulations.A total of 7279 positive urine culture results were collected from the laboratory information system between January 1, 2016, and Dec 31, 2019. The 1597 results in total of blood cell analysis, urine dipstick and urine sediment in 2019 were collected simultaneously. Considering the changes of bacterial virulence, we gathered the blood cell analysis, urine dipstick and urine sediment results within 2 days before or after the urine culture1 for a and b, UII2 for a to c, UII3 for a to d, UII4 for c and d, UII5 for c to f, and UII6 for a to f, to decide which one is more suitable to predict UTI.UII were calculated by the following formula: es Table  represenP < 0.05 was considered statistically significant.Normally distributed continuous variables were compared by t-test, while non-normally distributed variables were compared by Mann–Whitney U test. Correlation of the non-negative rate of LE and WBCs was calculated by Pearson’s correlation analysis. ROC curve analyses were performed, and the AUC was used to assess the performance of UIIs and SII. All analyses were performed in SPSS software version 24.0 and GraphPad Prism version 8. Supplementary Information


Poa pratensis L., and here we present a detailed work aimed at clarifying its role in apomixis. In situ hybridization showed that PpAPOSTART is expressed in reproductive tissues from pre-meiosis to embryo development. Interestingly, it is expressed early in few nucellar cells of apomictic individuals possibly switching from a somatic to a reproductive cell as in aposporic apomixis. Moreover, out of 13 APOSTART members, we identified one, APOSTART_6, as specifically expressed in flower tissue. APOSTART_6 also exhibited delayed expression in apomictic genotypes when compared with sexual types. Most importantly, the SCAR (Sequence Characterized Amplified Region) derived from the APOSTART_6 sequence completely co-segregated with apomixis.The production of seeds without sex is considered the holy grail of plant biology. The transfer of apomixis to various crop species has the potential to transform plant breeding, since it will allow new varieties to retain valuable traits thorough asexual reproduction. Therefore, a greater molecular understanding of apomixis is fundamental. In a previous work we identified a gene, namely APOSTART, that seemed to be involved in this asexual mode of reproduction, which is very common in  Angiosperms reproduce primarily through sexual reproduction, however in plants, this requires a series of developmental steps that culminate in the formation of the seed. Meiosis and fertilization as the main signs of sexuality, secure the formation of genetically variable diploid progeny . HoweverApomixis is a challenging trait that offers unique opportunities for developing superior, true breeding cultivars irrespective of their level of heterozygosity ,4,5. TheThe main advantages in having an apomictic genotype are that it reduces cost and breeding time for a new cultivar, avoiding various complications associated with sexual reproduction, such as incompatibility barriers and viral transfer in plants typically propagated vegetatively . MoreoveToday, the general consensus regarding apomixis and sexual reproduction indicates these as evolutionarily related processes, sharing many regulatory components. Once apomictic genes initiate embryo development and the initial cell forms and divides, the genes controlling embryo cell formation and patterning are most likely the same as those required for sexual embryo development. These similarities recently have been debated ,8,9, andPoa, Brachiaria, Cenchrus/Pennisetum, Panicum, and Paspalum. Among these, we elected to investigate Poa pratensis L. Kentucky bluegrass (P. pratensis) is a highly polyploid, hardy, persistent, and attractive forage and turf grass adapted to a wide range of soils and climates. Its mode of reproduction is extremely versatile ranging from sexuality to natural obligate apomixis (apospory and parthenogenesis). In P. pratensis, apospory involves the development of embryo sacs from somatic cells that differentiate into the nucellus. If unreduced polar nuclei positioned centrally within the embryo sac fuse with a sperm cell released from the pollen tube (pseudogamy), the unreduced egg can develop autonomously through parthenogenesis to form viable apomictic seeds [P. pratensis and isolated 2.248 ESTs, most of which (60%) were specific of floral organs and/or were involved in megasporogenesis and seed development. In particular, one expressed sequence tag (EST) clone showed a high similarity with an EST isolated from a pistil-specific cDNA library of apomictic Pennisetum ciliare and with the human StAR gene that can cause pseudohermaphroditism. Due to its START domain and putative involvement in apomixis, we named this gene APOSTART [APOSTART_1 and APOSTART_2). Here, we report the isolation of additional 13 cDNA and six genomic clones for which we determined genomic organization, temporal, and spatial expression and, computationally, infer on the binding of phytosterols at the START domain of their coded proteins. Moreover, we used the sequence of our best candidate (APOSTART_6) to search for similar proteins and found 207 proteins belonging to 158 species. Their relationships based on distances in the protein amino acid sequences composition were used to build a phylogenetic tree. The putative involvement of APOSTART in apomixis is also reported and discussed.Apomixis occurs in thousands of species across all kingdoms of the Eukaria , and thiic seeds . Moreoveic seeds . Therefoic seeds  applied APOSTART . In thisPoa pratensis were used to amplify, clone, and sequence the full-lengths of APOSTART members  and two highly apomictic genotypes (RS7-3 and L4) of  members .P. pratensis segregating F1 population (PG-F1) derived from a cross between S1/1-7 and RS7-3 [1 interspecific hybrids from a cross between a sexual P. arachnifera (Pa1FM) and a highly apomictic P. pratensis genotype, evaluated for their mode of reproduction by progeny tests   . These PP. pratensis (qPCR (quantitative PCR): S1/1-7 , RS7-3 and L4 (apomictic), together with three progeny plants from the S1/1-7 × RS7 cross  and four progeny plants from the S1/1-7 × L4 cross  of ratensis . Reprodu® Suite 8 Contig Express . Sequences were aligned using VECTOR NTI® Suite 8 AlignX software . Based on this alignment, specific primers were designed to perform end-to-end PCR on cDNA and genomic DNAs . After a first confirmation sequence was performed using M13 forward and reverse primers, a primer walking approach was adopted to sequence the complete clones. Alignment between the full-length cDNAs and genomic clones . The plasmid DNA was purified from 5 mL of an overnight culture on LB medium of c clones  disclosec clones .P. pratensis, were collected. Nucleic acids were isolated from about 0.1 g of fresh tissue using the GenElute total RNA purification kit , according to the manufacturer’s instructions, with some modifications to adapt it to plants. Total RNA was purified from residual genomic DNA by using the DNA-free . Reverse transcription and second-strand synthesis were carried out with 1 µg of total RNA, and the standard procedure was followed [Florets, harvested at five developmental stages  according to cytohistological investigations ,14, leavfollowed .t data from the MX3000P instrument were exported to a data file and analysed using GeneEx Pro software . During the pre-processing phase, data were corrected for PCR efficiencies and the three technical repeats were averaged. The selected reference genes, P. pratensis β-tubulin [t values [t value. Because our interest was in fold changes in gene expression between groups, we ultimately converted quantities to a logarithmic scale using a log base 2 conversion, which also allowed us to test the normal distribution of values.All qPCR analyses were performed using an Mx3000P qPCR  system with the SYBR green JumpStart Taq ReadyMix for quantitative PCR . Specific primers were designed within the sequences of each allele . The PCR-tubulin  subsequet values ,19, and ® Suite 8 AlignX software  to disclose differences in nucleotide composition. Several primer pairs were designed on the basis of genomic differences and tested on the F1 population segregating for the mode of reproduction . At each stage, single spikelets were collected, fixed in ethanol-formaldehyde-acetic acid, embedded in paraffin, and used for ISH experiments. Tissue preparation and hybridization conditions were the same as described by Angenent et al. [During inflorescence development, in sexual and apomictic t et al. .Sense and antisense probes were obtained by in vitro transcription using cloned PCR-derived fragments APOSTART as templates. In particular, APOSTART riboprobes were obtained from a single cDNA fragment of 1060 bp, which comprised only a small part of the START and DUF1336 domains and the sequence between the two domains. APOSTART riboprobes did not discriminate between alleles due to the very small differences in sequences.2CO3/NaHCO3 buffer, pH 10.2, for about 35 min. Immunological detection was performed as described by Cañas et al. [DIG-UTP sense and antisense riboprobes were synthesized by the T3 and T7 RNA polymerase. Transcripts were partially hydrolyzed by incubation at 60 °C in 0.2 M Nas et al. .APOSTART_6 cDNA and amino acid sequences as a query, similarities were searched in the National Center for Biotechnology Information  [Using nih.gov) . In bothnih.gov) . A discrnih.gov) .http://raptorx.uchicago.edu/) and PredictProtein servers (http://www.predictprotein.org) [Phosphorylation sites analyses of the 15 APOSTART amino acids were performed with NetPhos 3.1 Server . Secondaein.org) ,27,28,29ein.org) , DeepLocein.org) , MultiLoein.org) , Plant-mein.org) , and Sheein.org) , Yloc [3ein.org) .The protein sequences coded by APOSTART_1, APOSTART_6, and APOSTART_8 genes were subjected to the computational workflow depicted in −1 Å−1. A further refinement of the obtained protein structures of APOSTART_1 and APOSTART_6 was carried out by the use of the large-scale low-mode (LLMOD) algorithm implemented in MacroModel [−1 Å−1). Each LLMOD run provided 20 conformations that were further optimized with MacroModel, and the resulting lowest energy structure was eventually selected as the final 3D model of the considered protein .The 3D structures of APOSTART_1, APOSTART_6, and APOSTART_8 provided at the homology modeling stage were refined by using the Protein Preparation Wizard implemented in Maestro . This stcroModel ,43. This−1 Å−1.The 3D structures of stigmasterol, brassicasterol and campesterol were sketched in the Maestro workspace and the corresponding local minimum structures were calculated using the OPLS force field with the TNCG minimization algorithm and a convergence threshold of 0.05 kJ molSubsequently, docking calculations were performed by the use of Glide software  in two spKa [The bound complexes of APOSTART_1, APOSTART_6, and APOSTART_8 obtained from the docking calculations underwent a further refinement step consisting of a multiple minimization performed with MacroModel using the same settings as protein structure minimization see . The 90 pKa  and tautpKa , scaffolpKa , 3D-QSARpKa , and metpKa . Using tijS is the i-th scoring value of j-th scoring, and jσ is the standard deviation on the j-th set of N scoring values. The j-th normalized score is then obtained as the deviation of penalty from its maximum:The consensus scoring was obtained by averaging the estimations of Glide, MacroModel, and FLAP. For this purpose, each set of estimations was transformed to assign the same metrics, based on standard deviation units , to the three-scoring set. The deviation of each value from the minimum score  was calculated with:i-th bound system is eventually expressed by:The consensus scoring for the The bound complexes of APOSTART_1, APOSTART_6, and APOSTART_8 with stigmasterol, brassicasterol, and campesterol gaining the highest consensus scoring (two top-scored per bound complex) were further investigated by molecular dynamics (MD) simulations with the Gromacs package . Each boThe system configuration obtained at 500 ps of each production MD run (vide infra) was selected for a final consensus scoring of each ligand pose. For this purpose, only the water molecules within 5 Å from the center of mass of the bound ligand were included in the model, which then underwent a local minimization with MacroModel (see above). The minimized structures were thus processed using the same approach employed to estimate the ligand–protein affinities in the bound complexes obtained by docking, and, thus, providing a value of consensus scoring for each bound complex.Starting from the sequences reported in , forwardThe final length of the cDNA clones ranged from 2158 nt for APOSTART_14 to 2256 nt for APOSTART_15, while the total length of the eight genomic clones ranged from 5030 nt (APOSTART_6) to 5531 nt (APOSTART_8). The alignment between genomic and full-length cDNA clones confirmed what was already known for APOSTART_1 and APOSTART_2 , that alMoreover, the average identity of the 15 cDNA clones was 96.87% ranging from 91.48% to 100% . The mosExpression of six APOSTART members was assayed in genotypes with different modes of reproduction by using allele specific primers in the qPCR analyses . Allele Overall, three of the six members investigated  were expressed in floral tissues as well as in roots and leaves. In all three cases the higher expression was recorded in flower tissues at anthesis, while in leaves and roots it was, on average, lower than in flowers . The expWhile in sexual genotypes APOSTART_6 shows an increasing expression level from pre-meiosis to anthesis after which its expression drops to an undetectable level; in apomictic genotypes it is possible to note a shift in expression. In fact, the apomictic genotypes APOSTART_6 starts to be expressed at post-meiosis, and it remains expressed throughout meiosis until the post-anthesis stage.Since our population included one recombinant genotype (non-aposporic and parthenogenetic), we performed a new qPCR analysis for APOSTART_6 including this genotype. As shown in 1 population of 68 individuals segregating for the mode of reproduction [P. pratensis or Poa aracnifera × P. pratensis provided by the USDA, Grazinglands Research Laboratory, El Reno, OK, USA all having adaptation to the contrasting environments of Southern and Eastern Europe and the Southern Plains Region of the United States. The reproductive behavior, and the provenance of the analyzed genotypes, are reported in Based on the results of the qPCRs, we were interested in determining if APOSTART_6 co-segregated with apomixis. To do so, we aligned all APOSTART genomic clones  and desioduction ,15,62 anoduction ,15,62. Thttps://blast.ncbi.nlm.nih.gov/Blast.cgi) [Since APOSTART_6 completely co-segregated with apomixis, we searched in the BLAST database (ast.cgi)  for protPhyscomitrella patens and Marchantia polymorpha, two non-vascular plants belonging to Bryophyta and Marchantiophyta, respectively, clustered together in an outgroup clade. Genera with more than one sequence  exhibited consistent clustering. Poa sequences clustered together as well. The first main cluster in fact includes monocots, in particular the Poaceae family (in red). Continuing counterclockwise, monocots are followed by a few independent taxa  belonging to the Magnoliopsida class, intermediate between mono- and dicotyledons. The last large clade contains all the Eudicots class plants, divided into very homogeneous groups corresponding to the orders and then to the botanical families. As a matter of fact, it is possible to Solanales, divided in Solanaceae (in light blue) and Convolvulaceae, Lamiales with several families, Asteraceae (in green), Malvaceae (in yellow), Malpighiales with Salicaceae and Euphorbiaceae, Brassicales, divided in Brassicaceae to which Arabidopsis belongs (in purple), Cleomaceae and Caricaceae, Myrtales, Fagales, Cucurbitaceae, and Fabaceae (in dark blue).From a first look at the tree, Dichanthelium oligosanthes is not in the Poaceae cluster and Rosales (in black), with Rhamnaceae on one side and Rosaceae, Cannabaceae, and Moraceae on the other, clustered in independent clades, suggesting that APOSTART_6 phylogeny is strictly dependent on systematic botany.Overall, only one sequence of APOSTART expression was investigated by in situ hybridization (ISH) in longitudinal sections of sexual and apomictic flowers. In a previous analysis , APOSTARWe predicted the subcellular localizations of the 15 APOSTART proteins based on amino acid sequence motifs. We used six different software, and their outputs were not coincident. Despite this, it is evident that the most predicted localization for PpAPOSTART proteins is the mitochondrion and the nucleus .P. pratensis  distinguished APOSTART_13, APOSTART_14, and APOSTART_11 from the others. In the PCoA of threonines phosphorylation coordinate 1 (29.63%) separated 13 out of 15 APOSTART proteins into three groups  from whiP. pratensis APOSTART proteins and all APOSTART-like proteins (in monocots and eudicots) scored in the phylogenetic analysis were tested for protein structure prediction analyses.Sequences of all the proteins were also analyzed for secondary structure similarities. The secondary structure is defined by the pattern of hydrogen bonds of the protein, such as α-helices, β-sheets, and coils that are observed in an atomic-resolution structure. All the P. pratensis APOSTART protein structures. In addition, solvent accessibility was considered as another parameter to characterize the surface area of the protein. The relevance of this measure may explain how different protein shapes have different solvent accessibilities (distinct in exposed and buried sites). This may reveal that the examined genes do not share the same 3D protein structure, even with high sequence homology. PpAPOSTART proteins showed an average percentage of exposed sites of 48.13%, 23.5% of buried sites, and 26.6% of intermediate sites . IDP regions fail to form a stable structure and are characterized by a low content of bulky hydrophobic amino acids and a high proportion of polar and charged amino acids. This is usually referred to as low hydrophobicity . FurtherSeveral lines of evidence have indicated the role of START domain in the shuttling of steroids and other biological lipids between the mitochondria compartments and may suggest a possible relationship between the expression of APOSTART proteins and the trafficking of phytosterols.https://www.rcsb.org/) [To corroborate this hypothesis, the 3D structures of APOSTARTs START domains and their interaction with phytosterols, namely stigmasterol, brassicasterol, and campesterol, were modeled by using a computational workflow  based onsb.org/) , coded 1sb.org/) . Thus, 2sb.org/) . MD  in which the secondary structure was conserved in all-molecular systems to assay the effect of explicit solvation on the physterol binding properties. At this point, consensus scoring calculations were performed on the 18 sampled bound complexes yielding to the top scored poses of stigmasterol, brassicasterol, and campesterol at APOSTART_1, APOSTART_6, and APOSTART_8. The results of consensus scoring (performed as described in the previous section) are reported in As shown, ten of the 18 bound systems were characterized by score values higher than 2.3  and were identified as top-scoring and analyzed at major details. The poses of the investigated phytosterols binding at APOSTART_1 and APOSTART_6 is reported in It is worth noting that two binding site regions were eventually detected in the calculated molecular systems of either APOSTART_1 and APOSTART_6 . Four ofA successful transfer of apomixis to non-apomictics will allow clonal seed production in a myriad of crop species. Hopefully, this event will reduce input costs of fruit and vegetable production, while also raising yield . A betteStAR gene that is involved in human congenital lipoid adrenal hyperplasia. The clinical phenotype of this disease includes male pseudo hermaphroditism resulting from deficient fetal testicular testosterone synthesis. In apospory, a cell of the nucellus becomes an aposporous initial and then develops into a nonreduced embryo sac, which through parthenogenesis, gives rise to a viable embryo. How and why somatic cells of the ovule change their developmental fate and gain embryogenic potency is unknown. This question, together with the features of the StAR gene, made APOSTART our primary candidate for the control of apomixis. As a consequence, we decided to further characterize its expression, structure, and inheritance. Our data seem to confirm our vision. If we take all PpAPOSTARTs into consideration, we can see that while some of them are expressed in all tissues even if sometimes differentially between apomictic and sexual genotypes , APOSTART_6 is the only PpAPOSTART specifically expressed in tissue flowers. Moreover APOSTART_6 showed a delayed expression in apomictic genotypes when compared with sexual ones, and this tendency was confirmed in a parthenogenetic individual. Both sexual reproduction and apomixis involve life-cycle renewal from gamete or gamete-like cells following ploidy restitution. The difference in expression timing between apomictic and sexual genotypes may imply that APOSTART_6 is the key regulator that switches a “normal” nucellar cell into an aposporous initial.In a previous work  we have APOSTART is involved in the change of fate of these cells and, therefore, in embryo sac development from nucellar cells.In addition, as shown by ISH, in apomictic genotypes a strong signal was detected only in some nucellar cells neighboring the MMC. This confirmed the hypothesis that P. pratensis.Based on specific polymorphisms between PpAPOSTARTs, we developed some member-specific primer pairs, and the one designed for APOSTART_6 co-segregated with apomixis and was renamed APO-SCAR. This, together with the qPCR data, made APOSTART_6 the candidate to control apomixis in Moreover, an important quality of SCAR markers is their suitability on different genetic backgrounds. We then tested APO-SCAR on material belonging to different areas and continents and confirmed its capability to discriminate apomictic versus sexual genotypes. Therefore, APO-SCAR can be valuable tool for use in breeding and selection programs, considering that among the practical choices for minimizing time and costs of breeding programs, an easy method for early selection based on molecular markers would be of major advantage.Arabidopsis thaliana, known as AtAPOSTART_1 and AtEDR2 (or AtAPOSTART_2), that share a high amino acid similarity and identity score with PpAPOSTART proteins could be made. In fact, AtAPO1 protein has been predicted to be localized in mitochondria and plastids [The subcellular localization predicts that PpAPOSTART might be localized in the mitochondrion or nucleus, although the six different software programs we used provided contrasting results. Nevertheless, a comparison with two proteins belonging to plastids , while Vplastids  could noBoechera when pistils were subjected to variety of stress treatments, including carbohydrate starvation, osmotic stress, exposure to H2O2, and inhibition of brassinosteroid synthesis [EDR2, one of the Arabidopsis orthologs of PpAPOSTART, may play a role in lipid signaling, mitochondria, and the activation of PCD (Programmed Cell Death) in plants. The prediction of PpAPOSTART localizes in mitochondria matches with this hypothesis. We also suggested [P. pratensis may be related to the PCD that is involved in the non-functional megaspores and nucellar cell degeneration events that permit enlargement of maturing embryo sacs. The strong signal detected by ISH in degenerating cells of tetrads and in developing embryo sacs and nucellar cells in close proximity would appear to confirm this hypothesis.Conversions from diplospory to normal tetrad formation was observed in ynthesis . In addiynthesis . The conynthesis  that EDRuggested  that APOMoreover, even if our tests to identify phosphorylation sites are predictions, they provided interesting hints about the potential post-translational different functions of the 15 PpAPOSTART identified. In serine, threonine, and tyrosine PCoA some PpAPOSTART members showed a clear differentiation from all others and, in particular, APOSTART_6 was separated from other members in all tests. This suggests a potential unique post-translational molecular function, in line with its flower-specific expression, and could represent a sign of its specific, important role in the cell changing fate and/or the apomictic embryo sac development.Arabidopsis and rice.Due to cell proliferation during evolution, the START domain acquired two distinct functions: the first appears to be generally related to the stress response ,75,76 anSterols are essential membrane components and are critical for many physiological processes in all eukaryotes. In plants, it has been demonstrated that sexual reproduction is highly affected when the ratio of campesterol to sitosterol differs by a factor of 10, and yet vegetative growth such as stem elongation is only moderately affected when the ratio of campesterol to sitosterol differs by a factor of 30 to the wild-type . MoreoveOryza spp. sequences to PpAPOSTARTs, this makes APOSTART a gold candidate for future editing approaches for improving the maternal-like rate in rice progenies.Recently, using CRISPR-Cas9, some researchers attempted to set up synthetic apomixis in rice but a maximum of 29% of offspring were maternal clones ,81,82. H