Spodoptera frugiperda (J. E. Smith), is a typical invasive migratory pest with a strong reproductive capacity, which has caused serious damage to crops. Larvae of S. frugiperda exhibit high levels of cannibalism associated with larval crowding. However, the response of S. frugiperda adults to such stress remains unclear. In this study, we investigated the effects of larval density on flight and reproductive parameters of S. frugiperda adults. We found that S. frugiperda reared under high-larval density conditions exhibited lower pupal and body weights, weaker flight and reproductive capacities than those reared as solitary larvae. This result was different from that of many migratory insects, where larval crowding enhanced migratory propensity of subsequent adults. In contrast, high-larval density conditions did not increase adult migration propensity in S. frugiperda. These findings enhance our understanding of migratory and reproductive behaviors of S. frugiperda in response to larval density.The fall armyworm, Spodoptera frugiperda is lacking. In this study, flight and reproductive performances in response to larval crowding were investigated in S. frugiperda. We found that larval crowding significantly reduced the pupal and body weights of S. frugiperda. Adults reared under the highest density of 30 larvae/jar had the minimum wing expansion, which was significantly smaller than that of larvae reared under solitary conditions. Larval crowding also significantly increased the pre-oviposition period (POP) and period of first oviposition (PFO) but decreased the lifetime fecundity, flight duration and flight distance. Our results showed that S. frugiperda reared under solitary conditions exhibited higher pupal and body weights and stronger reproductive and flight capacities than those reared under high-density conditions. Larval crowding did not enhance the migration propensity in S. frugiperda adults. In conclusion, larval crowding may not be a major factor affecting FAW migration due to high levels of cannibalism. These findings provide new insights into the population dynamics of S. frugiperda under larval crowding conditions.Reproduction and flight are two major adaptive strategies to cope with environmental stress in migratory insects. However, research on density-mediated flight and reproduction in the global migratory agricultural pest  Larval density is one of the important abiotic factors shaping the behavior and life history of organisms. In holometabolous insects, larval crowding increases resource competition and the risk of cannibalism  and can thus affect individual fitness-related traits ,2,3, sucMythimna separata  [Loxostege sticticalis L. [Agrotis ipsilon (Hufnagel) [Cydia molesta (Busck) [Drosophila melanogaster (Guenee) and Tribolium castaneum (Herbst) [Mallada basalis , cannibalism can enhance the development and fecundity [Spodoptera exigua (Hübner) [Helicoverpaarmigera (Hübner) [Migration is usually considered an important life-history trait to cope with adverse environmental conditions . Indeed, ,18 and Lcalis L. . Howeverufnagel)  and Cydi (Busck) . Many st (Busck) ,22. In a(Herbst) ,24. Cann(Herbst) ,25. In Mecundity . Howeverecundity . In someecundity , such as(Hübner)  and Heli(Hübner) .Spodoptera frugiperda J. E. Smith) is a typical, migratory and destructive agricultural pest with a world-wide distribution. Due to its long-distance migration, high dispersion rate, generalist food habits and strong reproductive capacity, the FAW poses a serious threat to global food security [The fall armyworm  used in this study were collected from a maize field in Nanning, Guangxi Zhuang Autonomous Region, China , in May 2019 and reared in the laboratory for one generation before the initiation of the experiments. The larvae were supplied with fresh corn seedlings until pupation in a glass jar (850 mL). Pupae were placed on sterilized soil with 15% moisture. After eclosion, newly emerged adults were individually paired in cylindrical plastic cages (1000 mL) and provided with 10% honey water. This colony was maintained at 27 ± 1 °C and 60% ± 5% relative humidity, with a 16-h light: 8-h dark photo period.S. frugiperda [Newly hatched FAW larvae were reared at densities of 1, 5, 10, 20 and 30 larvae per 850 mL glass jar throughout the whole feeding period. Evidence of cannibalism associated with high larval density has been reported in ugiperda . We examS. frugiperda adults from five larval density treatments were anesthetized with ether, the body weights and wing expansion  of 1-d-old female and male adults after emergence were determined using electronic balance and electronic Vernier calliper , respectively. Sample sizes for body weight (from low to high rearing density) were 50, 34, 27, 36 and 38 females, and 51, 33, 52, 38 and 47 males, respectively. Sample sizes for wing expansion (from low to high rearing density) were 44, 30, 27, 35 and 36 females, and 53, 27, 50, 37 and 38 males, respectively.Pupal weight of 4-d-old pupae was measured using an electronic balance . The sample sizes for pupal weight from 1 to 30 larvae per jar were 71, 37, 65, 41 and 60, respectively. After weighing, the pupae were put back into the original glass jar until eclosion. When newly emerged S. frugiperda, reproductive parameters were measured using the method described in our previous studies [To explore the effects of larval density on adult reproduction in  studies ,47,48. A studies . The samFlight tests were conducted by using a 48-channel computerized flight mill system . Individp < 0.05). Pearson correlation analysis was conducted to examine the effects of body weight and wing expansion on reproductive and flight capacity in adults. All statistical analyses were performed using SPSS software .All data obtained in this study are presented as means ± standard errors. The effects of larval density on pupal weight, body weight, wing expansion, and reproductive and flight parameters were assessed with one-way analysis of variance followed by Tukey’s honestly significant difference (HSD) post hoc test . The weight of pupae reared as solitary larvae was significantly higher than that of pupae reared at densities of 10, 20 and 30 larvae per jar . With increasing larval density, the body weights of adults decreased continuously. The body weights of female and male adults reared solitarily were significantly higher than those of adults reared at densities ranging from 5 to 30 larvae per jar. The female and male adults from the 30 larvae per jar treatment group had the lowest body weights, which were significantly lower than those in the other treatment groups . The degree of wing expansion in females reared at a density of 30 larvae per jar was significantly lower than those in females reared at the other densities ; however, it did not differ from those of females in the other density treatments . Females from the solitary larva treatment group produced more eggs than adults from the other four larval treatment groups  or adult longevity . The flight distances of males reared at densities of 1, 5 and 10 larvae per jar were significantly longer than that of males reared at a density of 30 larvae per jar  [Sitophilus granarius L.) [S. frugiperda adults differed significantly under different larval-density conditions. The degree of wing expansion in FAW adults reared solitarily was significantly greater than that in those reared at a density of 30 larvae per jar. However, no differences were observed in L. sticticalis [L. migratoria [S. frugiperda.In our study, the pupal weight and body weight of separata , L. sticcticalis , Athetisoschler)  and otheoschler) ,51. The oschler) , and therius L.) . Previourius L.) ,43. We acticalis  and L. mgratoria . PearsonM. separata [S. exigua [L. sticticalis [L. sticticalis and M. separata, moderate larval crowding triggers migratory behavior, but overcrowding decreases the migration propensity because physiological stress reduces flight capacity [C. medinalis adults is not enhanced under crowding conditions during the larval phase [H. armigera, high larval density also does not increase their flight capacity [S. frugiperda in both sexes, resulting in solitary FAWs having a better flight capacity than those reared at higher larval densities. This result was consistent with some previous reports that high larval density did not produce pre-migrant traits, as larval density had little effect on developmental time, adult activity, whole-body weight and lipid contents in FAW [S. frugiperda larvae. Previous studies found that the distribution pattern of S. frugiperda larvae was closely associated with cannibalism [S. frugiperda between the first and third instar showed a pattern of aggregation distribution, but the later larvae exhibited an even distribution in maize and wheat fields [S. frugiperda. Wang et al. [S. frugiperda larvae mainly occurred at the fifth instar stage. Cannibalism can reduce competition and obtain additional high-value nutritional resources while decreasing search time [S. frugiperda survival under starvation and successfully colonize new food plants [S. frugiperda individuals reared under larval crowding conditions, resulting in a decreased flight potential and flight capacity of subsequent adults.Environment-dependent polyphenism of reproduction and flight are major adaptive strategies that strengthen the ecological success of many migratory insects ,55,56,57separata ,18, S. e. exigua  and L. scticalis . Migrantcticalis . In L. scapacity ,10. Howeal phase . In H. acapacity . Our ress in FAW ,62. Our nibalism ,64,65. Lt fields ,64. He et fields  reportedg et al.  found thrch time ,66, whicd plants . We thusMythimna convecta [M. separata [S. exigua [L. sticticalis [C. medinalis [S. frugiperda adults, resulting in a delay of first oviposition and decreases in synchronized oviposition and lifetime fecundity. These results indicate that FAWs reared solitarily as larvae exhibit a stronger reproductive performance. Furthermore, a negative correlation between larval density and reproduction also existed in S. frugiperda. Thus, larval density was one of the important factors affecting adult migration in many migratory insects without cannibalism or with low levels of cannibalism. Whereas, for those insects with high levels of cannibalism, larval crowding aggravated internal competition and cannibalism, which resulted in increased mortality, delayed development time and decreased adult reproductive and flight capacity. We speculated that larval crowding would not be the main factor inducing adult migration in FAW, owing to no significant enhancement of adult migration propensity and capacity for adult migration. Therefore, more attention should be paid to the effects of temperature, wind, photoperiod and other weather factors on reproduction and migration to better predict outbreaks in FAW.Crowded conditions reportedly decrease the reproductive performance of many insects, such as convecta , M. sepaseparata ,58, S. e. exigua , L. sticcticalis  and C. medinalis . Our resS. frugiperda, which may also be a widespread adaptive strategy in many migratory insects to cope with environmental stressors during the larval phase. FAWs reared solitarily exhibited higher pupal and body weights and stronger reproductive and flight capacities than those reared at higher larval densities. Larval crowding did not produce pre-migrant traits and enhance the migration propensity in S. frugiperda adults. Therefore, larval crowding may not be a key factor in inducing the migratory behavior in S. frugiperda in the case of high levels of cannibalism. These findings may enhance our understanding of the adaptive behavioral strategies of S. frugiperda in response to unfavorable conditions and provide novel strategies for the control of this invasive pest.In conclusion, our results suggest that alterations in flight and reproductive traits may be strongly dependent on the level of larval crowding in


Oxidative stress (OS) disrupts the chemical integrity of macromolecules and increases the risk of neurodegenerative diseases. Fisetin is a flavonoid that exhibits potent antioxidant properties and protects the cells against OS. We have viewed the NCBI database, PubMed, Science Direct (Elsevier), Springer-Nature, ResearchGate, and Google Scholar databases to search and collect relevant articles during the preparation of this review. The search keywords are OS, neurodegenerative diseases, fisetin, etc. High level of ROS in the brain tissue decreases ATP levels, and mitochondrial membrane potential and induces lipid peroxidation, chronic inflammation, DNA damage, and apoptosis. The subsequent results are various neuronal diseases. Fisetin is a polyphenolic compound, commonly present in dietary ingredients. The antioxidant properties of this flavonoid diminish oxidative stress, ROS production, neurotoxicity, neuro-inflammation, and neurological disorders. Moreover, it maintains the redox profiles, and mitochondrial functions and inhibits NO production. At the molecular level, fisetin regulates the activity of PI3K/Akt, Nrf2, NF-κB, protein kinase C, and MAPK pathways to prevent OS, inflammatory response, and cytotoxicity. The antioxidant properties of fisetin protect the neural cells from inflammation and apoptotic degeneration. Thus, it can be used in the prevention of neurodegenerative disorders.  Neurodegenerative diseases are characterized by the permanent loss of a specific type of neurons in the brain, leading to progressive loss of brain functions. The subcortical area of the brain particularly basal ganglia is mostly affected in certain neurodegenerative diseases. Elderly people are vulnerable to neurodegenerative diseases . The mosMitochondrial dysfunction and oxidative stress play crucial roles in the development of neurodegenerative diseases. Mitochondria are the main site for metabolic reactions where electrons are finally transported through the electron transport chain (ETC). Any impairment in mitochondrial function increases the production of reactive oxygen species like superoxide anion radicals (O−2) . MitochoApoptosis and ferroptosis of neurons is an important features in neurodegenerative disorders. Human postmortem brain samples showed detectable DNA fragmentation in PD, AD, HD, and ALS. Moreover, the presence of caspases and BclXL is also associated with Alzheimer’s disease. Different caspases are involved in apoptosis. Caspase-6 plays an important role in the pathogenesis of AD and HD, whereas caspase-7 is activated during motor neuron degeneration in a mouse model of ALS. Thus, apoptosis causes chronic neurodegeneration and neuron loss. Abnormal expression of cell cycle regulator proteins has also been implicated in neurodegenerative diseases. Additionally, increased levels of p53 in motor neurons of the spinal cord and motor cortex induce neuronal degeneration .Basal ganglia are situated bilaterally in the white matter of the cerebral hemispheres and comprise five subcortical nuclei. These include the caudate nucleus, putamen, subthalamic nucleus, substantia nigra (SN), and the globus pallidus (GP). SN has two parts: 1) SN pars compacta and 2) SN reticularis/reticulata. On the other hand, GP has two segments: 1) GP internal and 2) GP external . The cauThe efferent connections from basal ganglia terminate to the prefrontal and premotor cortex and the ventral lateral, ventral anterior, and centromedian nuclei of the thalamus via the thalamic fasciculus. SN projects connections to the other areas of the thalamus. Additionally, basal ganglia are associated with other nuclei in the diencephalons and mid-brain. Basal ganglia are mainly involved in the regulation of activity of the motor area of the cerebral cortex for controlling movements. This regulatory function occurs through the way of the thalamus. Basal ganglia also control cognitive functions.The neurons of the pars compacta of SN are dopaminergic. The nigrostriatal pathway releases dopamine as a neurotransmitter in the striatum, particularly in the putamen. Dopamine acts as an excitatory neurotransmitter to modulate the activity of inhibitory pathways (GABAergic) from the striatum  to the internal segment of the GP  Ehrlich. HoweverFisetin, chemically 3,7,3′,4′-tetrahydroxyflavone or 7,3′,4′-flavon-3-ol is a polyhydroxy flavonoid with various pharmacological potentials like antioxidant , anti-inin vitro  in mice, its maximum plasma concentration was 2.5 μg/ml at 15 min and it was decreased biphasically with a half-life of 0.09 h; three metabolites of fisetin were detected in this study including two glucuronides and a methoxylated metabolite as 3,4′,7-trihydroxy-3′-methoxyflavone  . Geraldoin vitro ; also itin vitro . More, iin vitro . As a main vitro .clinicaltrials.gov) the oral dose of 20 mg/kg/day for two consecutive days and/or for two consecutive months are mostly used  as therapeutic agents to enhance the cognitive manifestations of AD without altering the progression of the disease pathology. In addition, research is being progressed to find the effective drugs to prevent its onset and progression . NumerouChronic inflammation increases the risk of neuronal damage. Fisetin induces the expression of tropomyosin-related kinase B (TrkB), brain-derived-neurotropic factor, PI3K-Akt, and ERK signaling pathways and inhibits the synthesis of inflammatory agents during the treatment of depression . FisetinNeuroprotective effects of fisetin in different neurological disorders have been indicated in several studies. The reviewed studies have been discussed based on each disease including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Anxiety and depression, Amyotrophic Lateral Sclerosis (ALS), Multiple sclerosis, Age-related changes, Neuropsychiatric disorders, Vascular dementia and Cognitive dysfunction which are shown by detail in vitro conditions. To investigate the structural requirements of fisetin for its anti-amyloidogenic activity, Aβ1-42 (20 µM) and several structurally related flavonoids were incubated, after that fibril formation was quantitatively determined. Fisetin was among the flavonoids that inhibited the formation of Aβ fibril; the 3′, 4′-dihydroxyl group was suggested to play an important role in its inhibitory effect  . It significantly decreased Aβ aggregation and modulated the expressions of ASK-1, p-JNK, p53, cytochrome c, caspase-9 and 3 as well as Bax/Bcl-2 ratio in cortex and hippocampus of mice administered AlCl3. AlCl3-induced neurodegeneration in the cortex and hippocampus also decreased via fisetin as observed by TUNEL and fluoro-jade C staining  and decreased the cytotoxic effect of LPS-stimulated microglia on B35 neuroblastoma cells , fisetin, as a positive control, exhibited protection in several assays reflecting multiple, age-associated neurotoxicity/survival mechanisms related to Alzheimer’s disease, such as elevated oxidative stress and GSH depletion, decreased energy metabolism, increase of aggregated proteins, inflammation and loss of neurotrophic support (Alzheimer’s disease (AD) is a deadly progressive neurodegenerative disorder and the most common type of irreversible dementia . Fundamey effect . Althougy effect . Some ot; i. p.) . Treatme; i. p.) . Insolub; i. p.) . In vitron model . It is kon model . As downon model . In Aβ1–on model . Furtherstaining . Fisetinstaining . In micestaining . Curraisstaining . It prevstaining . Oral adstaining . In a ststaining . Followistaining . It has staining . In 2009staining . Fisetinstaining . Cholinestaining  and the staining . The anistaining ; memory staining . Fisetin and SOD . Its inhin vitro . Accordiin vitro . Mild hyin vitro . Anotherin vitro . Fisetinin vitro . Moreovein vitro . In vitrma cells . As a mema cells . Adminisma cells . In an ineration . Fisetinactivity . In a st support . The act25-35-induced neurodegeneration.Parkinson’s disease (PD) is a neurodegenerative disease , charactHD. Normally, this gene contains 11–34 (with a median of 19) glutamine-specific codon (CAG), but in the disease state this number increases up to 37–86 (with a median of 45) or more. The gene HD encodes a protein designated huntingtin, which is involved in the regulation of the transcription of neurotransmitter receptors in the brain. In the basal ganglia, huntingtin plays a vital role in transcriptional processes as well as axonal transport and membrane trafficking. The mutant form of huntingtin cannot regulate these functions and its accumulation promotes the risk of disease. Mutant huntingtin protein (Htt) is a misfolded soluble protein that contains a polyglutamine (poly Q) stretch. Htt-25Q is non-toxic, while Htt-103Q formed soluble aggregates and cytotoxicity . R6/2 mice show aggressive disease phenotype and shortened lifespan. Feeding of fisetin-containing diet (∼45 mg of fisetin/kg bw/day) from the age of ∼6 weeks increased motor activity (tested on the rotarod from ∼7 weeks) compared to control group of R6/2 mice consuming fisetin-free diet. The rate of survival of R6/2 mice taking the fisetin-containing diet was ∼30% higher (139 days) compared to the control group (104 days)  (Httex1p Q93) enhanced ERK phosphorylation and activation, resulting in ∼25% diminution of neurodegeneration, suppression of symptoms of HD and increased 77% of overall survival . This gene is also called toxicity . Increastoxicity . Experimtoxicity . Furthertoxicity . Glutamatoxicity . Moreovetoxicity . Zhao ettoxicity  reportedtoxicity . Experimtoxicity . These atoxicity . In vivo04 days) . The effsurvival . Fisetinsurvival .ALS is an age-related neurodegenerative disorder of motor neurons in the spinal cord, motor cortex and brain stem. The exact cause of ALS is still unclear. It has been assumed that glutamate-induced excitotoxicity is one of the possible causes of pathogenesis. Excess glutamate in the synapse or extracellular space is transported into neuronal and glial cells via glutamate transporter 1 (GLT-1) . Any mutDepression and anxiety are considered as the psychological symptoms of stress which are highly comorbid disorders. These conditions are important factors upsetting mental health that affects the individual’s life. Depression is known as one of the most common psychiatric conditions and suicide risk factors, which results in the decrease of quality of life, increase in drug consumption, healthcare costs, and other social and economic problems . DepressNatural products are being used as alternatives to pharmacotherapy for the management of various diseases including depression and anxiety . Among tMultiple sclerosis: Multiple sclerosis (MS) is a potential disease of the brain and spinal cord. It is a neuroinflammatory disease. The immune system attacks the myelin sheath on the neurons and causes permanent damage. The primary symptom is the inability to move. In MS demyelination starts when macrophages phagocytose myelin and produce inflammatory mediators. Fisetin inhibits in vitro myelin phagocytosis by decreasing the activity of macrophages. It potentially regulates the secretion of inflammatory mediators, chronic inflammation in the central nervous system and neurological deficits (2O2 and OH•) production without affecting the viability of macrophage cells. It also suppresses ROS-producing enzymes mainly xanthine oxidase, NADPH oxidase, etc. This plant derivative specifically decreases NF-κB-induced proinflammatory mediators like NO, IL-1, and TNF-α   is the imbalance of production, accumulation, and detoxification of ROS/RNS inside cells and even in tissues . The incβ . In AD pβ . ROS plaβ . Moreovese (Nox) . The patse (Nox) . The NADse (Nox) . Moreovese (Nox) . An excese (Nox) . Oxidatise (Nox) . OS strese (Nox) . It is ase (Nox) , which ese (Nox) . In 2016se (Nox) . Pitts ese (Nox) . Moreovese (Nox) . In PD, epletion . Moreoveepletion . Meiser epletion . Furtherepletion . The rotepletion . It is eepletion . About 2epletion . SOD1 scepletion . The SODepletion . Interesepletion . Besidesepletion . The oxiepletion . In MS, epletion . Severalepletion . Moreoveepletion . There iepletion . Some stpatients .drosophila , HO-1, and Glutathione S-transferase (GST) and even phase II detoxifying enzymes (A common dietary flavonoid fisetin, recently gained much scientific consideration for its antioxidant properties to inhibit a wide range of life-threating diseases . Antioxiosophila . Moreoveosophila . Besidesosophila . Wang etosophila . Besidesosophila . In a prosophila . In oxidosophila . The celosophila . In 2001osophila . Kang et by H2O2 . In anot by H2O2 . Tingtin by H2O2 . In addixidation . Chuang xidation . Recentlxidation . Xu et axidation . Howeverxidation , antioxixidation , and neuxidation  activityd center . Fisetind center . In partn others . Besidesn others . Moreoven others . Besidesn others . Lower Nn others . Sakai e enzymes . Moreove enzymes . Léotoin enzymes . P Maher enzymes  HT22 cel enzymes , and pri enzymes . The fis enzymes . Nrf2 in enzymes . These c enzymes . So, it  enzymes .Neurological disorders are a serious problem throughout the world and there is no definite treatment for this purpose. Excess production of ROS and poor detoxification process increase OS. Neurons are the most vulnerable to OS as they consume a greater amount of oxygen, contain much amount of polyunsaturated fatty acids in their membrane, and fail to maintain redox homeostasis. OS starts lipid peroxidation, protein carbonyl formation, DNA damage, induction of neuroinflammatory response, and apoptotic activity. Collectively, these responses promote the degeneration of neurons in the CNS. Fisetin belongs to flavonoid groups, scavenges ROS, and restricts OS. It also increases the expression of SOD, catalase, GPx, HO-1. Several studies indicate that fisetin is effective against AD, PD, HD, MS, and ALS. The multifaceted actions of fisetin increase the possibilities of its use to prevent neurodegenerative diseases. Finally, it can say that extensive research on the application of fisetin against CNS disorders and proper clinical trials will allow counteracting the neurological disorders.


The present systematic review investigated possible factors which may influence the surgical outcome of minimally invasive surgery for total hip arthroplasty (MIS THA).In January 2022, the Embase, Google Scholar, PubMed, and Scopus databases were accessed. All the clinical trials investigating the clinical outcome of MIS THA were considered.P = 0.02) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) (P = 0.009) at last follow-up, and shorter surgical duration (P = 0.01). Greater body mass index (BMI) at baseline was moderately associated with greater cup anteversion (P = 0.0009), Oxford Hip Score (OHS) at last follow-up (P = 0.04), longer surgical duration (P = 0.04), increased leg length discrepancy (P = 0.02), and greater rate of infection (P = 0.04). Greater VAS at baseline was weakly associated with greater VAS at last follow-up (P < 0.0001), total estimated blood lost (P = 0.01), and lower value of Harris Hip Score (HHS) (P = 0.0005). Greater OHS at baseline was associated with greater post-operative VAS (P = 0.01). Greater WOMAC at baseline was associated with lower cup anteversion (P = 0.009) and greater VAS (P = 0.02). Greater HHS at baseline was associated with shorter hospitalisation (P = 0.001).Data from 9486 procedures were collected. Older age was moderately associated with greater Visual Analogue Scale (VAS) (Older age and greater BMI may represent negative prognostic factors for MIS THA. The clinical outcome is strongly influenced by the preoperative status of patients. Minimally invasive surgery (MIS) for total hip arthroplasty (THA) has become popular . The defP (Population): end-stage OA;I (Intervention): MIS THA;C (Comparison): patients characteristics at admission;O (Outcomes): PROMs, radiological findings, complications;This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The PIChip, total, arthroplasty, replacement, prosthesis, instrumentation, surgery, intervention, BMI, age, sex. The resulting abstracts were screened by the two authors and, if of interest, the full-text was accessed. The bibliographies were also screened by hand. Disagreement was debated and solved by the senior author (N.M.).Two authors (F.M. and A.P.) independently performed the literature search in January 2022 accessing the following databases: PubMed, Google Scholar, Embase, and Scopus. The following keywords were used and combined for the search: All the clinical trials investigating the outcomes of MIS THA were accessed. Only studies comparing traditional versus MIS THA approaches were considered eligible. Given the authors languages capabilities, articles in English, German, Italian, French and Spanish were eligible. Only levels I to III of evidence, according to the Oxford Centre of Evidence-Based Medicine , were elPatient demographics: number of procedures, mean BMI and age, percentage of female;PROMs: Visual Analogue Scale (VAS), Oxford Hip Score (OHS), The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Harris Hip Score (HHS).The present study investigated whether the aforementioned endpoints were associated with the outcome. Thus, every single endpoint was independently analysed, and its association with the following data at last follow-up assessed:Peri-operative data: surgical duration, total estimated blood loss, and length of hospital stay;Radiographic measures: mean cup inclination and anteversion, mean stem alignment, and limb length discrepancy;PROMs: Visual Analogue Scale (VAS), Oxford Hip Score (OHS), The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Harris Hip Score (HHS);Complications: dislocations, revisions, deep infections, aseptic loosening, fractures.Two authors (F.M. and A.P.) independently performed data extraction. Study generalities  were collected. Data concerning the following endpoints at baseline were collected:The methodological quality assessment was made by two independent reviewers (F.M. and A.P.). The risk of bias graph tool of the Review Manager Software 5.3  was used. The following risk of bias was assessed for each included study: selection, detection, attrition, reporting, and other source of bias.χ2 test, with values of P > 0.05 considered statistically significant.The statistical analyses were performed by the main author (F.M.). For the analytical statistics, STATA MP 16 software  was used. The Shapiro–Wilk test was performed to investigate data distribution. For normal data, mean and standard deviation (SD) were calculated. For nonparametric data, median and interquartile range (IQR) were calculated. A multivariate analysis was performed through a multiple pairwise correlations according to the Pearson product-moment correlation coefficient The literature search resulted in 684 articles. Of them, 277 were excluded because they were duplicates. A further 297 articles were excluded since they did not match our eligibility criteria. Another 36 articles were not included because they did not report quantitative data under the outcomes of interest. This left 74 studies for the present study: 33 randomised, 29 prospective, and 17 retrospective studies. The literature search results are shown in Fig. The risk of bias summary evidenced some limitations of the present study. Approximately half of the studies were randomised, and approximately one fifth were retrospective. This leads to a moderate risk of selection bias. Given the overall lack of blinding, the risk of detection bias was moderate-high. The authors' judgements about the risk of attrition, reporting and other bias presented across all included studies was moderate. Concluding, the overall risk of bias was moderate, attesting to this study good quality assessment Fig. .Fig. 2Me2. Generalities and patient baseline of the included studies are shown in Table Data from 9626 procedures were collected. 57% (5487 of 9626 patients) were women. The median follow-up was 12 (IQR 9) months. The mean age was 63.0 (SD 4.9), the mean BMI 27.1 (SD 2.3) kg/mr =  − 0.52; P = 0.0002). Older age was moderately associated with reduced surgical time , and with greater VAS  and WOMAC scores  at last follow-up. Greater BMI at baseline was associated with greater cup anteversion , greater OHS at last follow-up , longer surgical duration , greater leg length discrepancy , and greater rate of deep infection . Greater VAS at baseline was associated with greater VAS at last follow-up , greater overall estimated blood lost , and lower value of HHS . Greater OHS at baseline was associated with post-operative greater VAS . Greater WOMAC at baseline was associated with lower cup anteversion  and greater VAS at last follow-up . Greater HHS at baseline was associated with shorter hospitalisation . No other statically significant associations were evidenced. The results of the multivariate analyses are shown in greater detail in Table Female gender was strongly associated with lower cup anteversion  were excluded for redundancy. To improve data pooling, both prospective and retrospective studies were included in the analysis, which inevitably increases the risk of selection bias. A limitation of this study is represented by the relative short length of the mean follow-up. Half of studies were randomised, but, given the overall lack of blinding methods, the risk of detection bias was moderate-high. Furthermore, the different approaches for THA were not considered separately, nor were the different implant designs –81. GiveOlder age and greater BMI were negative prognostic factors for MIS THA. The analyses of the PROMs suggested that the clinical outcome is strongly related to the preoperative performance status of the operated patients. There is no compelling evidence that MIS THA offers advantages over traditional approaches, especially when modern analgesia techniques and accelerated rehabilitation programmes are considered.


Background: Helicobacter pylori gastritis affects two-thirds of the world's human population. Among various invasive and non-invasive tests, histology play a very important role in detecting H. pylori in gastric biopsies. In histology, for detection of H. pylori, we use different histological staining techniques like routine haematoxylin & eosin (H&E) stain, Giemsa stain, Gimenez stain, and periodic acid Schiff - Alcian blue (PAS-AB) stain.Objective: Aim of our study was to evaluate these different histopathological staining techniques for detecting H. pylori in gastric mucosal biopsies and to determine the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of H&E stain, Gemenez stain and PAS-AB in the detection of H. pylori in gastric biopsies using Giemsa stain as the reference standard.Materials and Methods: It was a prospective descriptive study design of 45 gastric biopsies of patients having gastritis. This study was conducted at Vivekananda Polyclinic and Institute of Medical Sciences over a period of one year, from March 2021 to February 2022. From each formalin-fixed paraffin-embedded block, four glass slides were prepared and stained with H&E stain, Giemsa stains, Gemenez stain, and PAS-AB stain to detect the presence/absence of H. pylori in gastric biopsies. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were assessed.Results: Various staining techniques for detecting H. pylori in gastric mucosal biopsies were compared. In reference to Giemsa stain results, statistical analysis indicates that the diagnostic accuracy of the Gimenez stain, H&E stain, and PAS-AB stain were 95.6%, 91.1%, and 84.4% respectively. Gimenez stain is confirmed to be better than H&E stain and PAS-AB stain to detect H. pylori in 45 gastric biopsies of patients having gastritis. PAS-AB stain is the worst stain to detect H. pylori in gastric biopsies.Conclusion: Gimenez stain has higher diagnostic accuracy than PAS-AB stain in the detection of H. pylori in gastric biopsy. In fact, Gimenez stain has high sensitivity, specificity and diagnostic accuracy as compared to traditional H&E stain while PAS-AB stain has lower sensitivity, specificity and diagnostic accuracy. Thus, Gimenez stain is also recommended for the detection of H. pylori in gastric biopsy. Helicobacter pylori is the most common human pathogen and affects various gastroduodenal diseases . It is rThis was a prospective descriptive study that analysed 45 endoscopic gastric biopsies of patients having gastritis or/and gastric ulcer. This study was conducted in the pathology department of our institution, Vivekananda Polyclinic and Institute of Medical Sciences, over a period of one year, from March 2021 to February 2022. Ethical approval from the institutional ethical committee was taken along with informed consent obtained from each patient . Inclusion criterion was the presence of gastritis. Exclusion criteria were cases with dysplasia and carcinoma of stomach. Forty-five formalin-fixed, paraffin-embedded blocks of gastric biopsies were collected. After that four glass slides were prepared from each paraffin-embedded block of gastric biopsies. Then these glass slides were stained with H&E stain, Giemsa stain, Gimenez stain and PAS-AB stain, according to standard protocols. For control of the study two pathologists examined all the sections of H&E stain, Giemsa stain, Gimenez stain, and PAS-AB stain in combination for each case to detect the presence/absence of H. pylori through a microscope under oil immersion field. These slides were examined for the presence of H. pylori mainly in the mucus and gastric pits. After that data were collected in the form of H. pylori-positive gastritis and H. pylori-negative gastritis according to different types of stains. Giemsa stain results were used as reference for the calculation of true positive, true negative, false positive, and false negative values for other histopathological stains . Then we performed statistical analysis in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy (DA) of the H&E stain, Gimenez stain and PAS-AB stain.A total of 45 patients who had gastritis or/and gastric ulcer, were participated in this prospective descriptive study. Range of age of patients included in the study was from 19 to 80 years. Twenty-three patients were female and 22 patients were male. Different histological stains are used for the direct detection of H. pylori in gastric biopsies. These stains include H&E stain, Giemsa stain, Gimenez stain and PAS-AB stain. The spiral-shaped H. pylori organism can be directly identified in high magnification with oil immersion field by microscope , PAS-AB stain is the worst stain for detection of H. pylori in gastric biopsies with lower diagnostic accuracy test that is 84.4% in comparison to Gimenez stain that is 95.6%.Half of the world's human population is infected with H. pylori. It is the most common gastric pathogen that colonizes the mucous lining of the stomach . H. pyloDetection of H. pylori through various tests includes invasive and non-invasive tests. Invasive tests include histology, rapid urease test (RUT), culture, and polymerase chain reaction (PCR). Non-invasive test include urea breath test (UBT), stool antigen test, and serology ,9,12. InSeveral histopathological staining panels have been used for detection of H. pylori in gastric biopsies . H&E staThe data from this study showed that diagnostic accuracy of the Gimenez stain was higher as compared to H&E stain Table  which meSensitivity and specificity of H&E stain, Gimenez stain and PAS-AB stain in the detection of H. pylori are shown in Table PPV is the proportion of patients with a positive test who have the disease, while NPV is the proportion of patients with a negative test who do not have the disease . In our According to our study, Gimenez stain has high diagnostic accuracy than H&E and PAS-AB stain in the detection of H. pylori in gastric biopsies. Gimenez stain has high sensitivity, specificity as compared to traditional H&E stain while PAS-AB stain has lower sensitivity, specificity and diagnostic accuracy This study strongly recommends Gimenez stain and Giemsa stain used as additional special stain along with H&E stain for detection of H. pylori in gastric biopsies.


Background:CD161 has been identified as a prognostic biomarker in many neoplasms, but its role in breast cancer (BC) has not been fully explained. We aimed to investigate the molecular mechanism and prognostic value of CD161 in BC.Methods:CD161 expression profile was extracted from TIMER, Oncomine, UALCAN databases, and verified by the Gene Expression Omnibus (GEO) database and quantitative real-time polymerase chain reaction (qRT-PCR). The prognostic value of CD161 was assessed via GEPIA, Kaplan–Meier plotter and PrognoScan databases. The Cox regression and nomogram analyses were conducted to further validate the association between CD161 expression and survival. Gene set enrichment analysis (GSEA), Gene Ontology (GO) analysis, and KEGG pathway enrichment analysis were performed to probe the tumor-associated annotations of CD161. CIBERSORT and ssGSEA were employed to investigate the correlation between CD161 expression and immune cell infiltration in BC, and the result was verified by TIMER and TISIDB.Results: Multiple BC cohorts showed that CD161 expression was decreased in BC, and a high CD161 expression was associated with a preferable prognosis. Therefore, we identified the combined model including CD161, age and PR status to predict the survival (C index = 0.78) of BC patients. Functional enrichment analysis indicated that CD161 and its co-expressed genes were closely related to several cancerous and immune signaling pathways, suggesting its involvement in immune response during cancer development. Moreover, immune infiltration analysis revealed that CD161 expression was correlated with immune infiltration.Conclusion: Collectively, our findings revealed that CD161 may serve as a potential biomarker for favorable prognosis and a promising immune therapeutic target in BC. Then, we verified the findings by quantitative real-time polymerase chain reaction (qRT-PCR), using paired tissue samples from our center. Next, we assessed the relationship between CD161 expression and clinicopathological parameters of BC. Furthermore, we investigated the prognostic value of CD161 for BC, and predicted BC survival using a nomogram constructed with the independent prognostic factors derived from multivariate Cox regression analysis. Besides, we conducted functional enrichment analyses to probe the tumor-associated annotations of CD161. Finally, we analyzed the relationship between CD161 expression and immune infiltration. Our research indicated that CD161 could serve as a potential prognostic biomarker and immune therapeutic target for BC.Breast cancer (BC) is the most common cancer in females worldwide. Despite great advances in the diagnosis and treatment, BC relapses in a considerable number of patients. BC still accounts for 15.5% of all female cancer deaths . Currentetc. . Crosstaetc. . TILs haetc. . Previouetc. . Howeveretc. . Accordi T cells . It was  T cells . CD161 cmulation . Thereforognosis . In addirognosis . A recenor cells , suggestlater™ solution , immediately frozen in liquid nitrogen and storage at −80°C until further use for qRT-PCR. The use of human tissues in this study was approved by the Research Ethics Committee and all patients enrolled signed the informed consent.A total of 1097 BC patients from the TCGA database were enrolled. Included were patients with  a primar® III first Strand cDNA Synthesis Kit (+gDNA wiper) . Next, qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix . The primers used were as follows: human CD161 -Forward: 5′-AAACAACAGAGAGACCGGGT-3′; human CD161 -Reverse: 5′-TCCAAGGGTTGACAGTGTGAT-3′; human GAPDH-Forward: 5′-GGAGTCCACTGGCGTCTTCA-3′; human GAPDH-Reverse, 5′-GTCATGAGTCCTTCCACGATACC-3′. Primers were synthesized by the GenScript Company.Total RNA was extracted from the paired breast tumors and adjacent tissue samples by Olasma Kit . cDNA was prepared through The HiScriptCD161 in BC was investigated by TIMER (https://cistrome.shinyapps.io/timer/), Oncomine (http://www.oncomine.org), UALCAN , and validated by GSE10797 from GEO database and qRT-PCR of paired tissue samples. The binary logistic model was employed to explore the association between CD161 expression and clinicopathologic features, including stage, age, T, N, M, ER, PR, menopause and anatomic neoplasm subdivision downloaded from TCGA database.The expression of http://gepia.cancer-pku.cn/), Kaplan–Meier plotter  and PrognoScan (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html) were employed to evaluate the prognostic value of CD161 in BC, based on the data about mRNA expression and survival. Overall survival (OS) referred to the time from histological diagnosis to death or the last follow-up. Disease-free survival (DFS) was the time from histological diagnosis to disease progression, death, or last follow-up. Disease-specific survival (DSS) was defined as the time from histological diagnosis to death from BC. Besides, we conducted univariate and multivariate Cox analyses of BC data from TCGA. A number of variables were assessed to identify the independent prognostic factors, including stage, age, tumor size, lymph node status, distant metastasis, CD161 expression, ER status and PR status.The GEPIA  < 0.05 were considered remarkedly enriched. cBioPortal for Cancer Genomics  was used to identify the genes co-expressing with CD161. Then, we performed Gene Ontology (GO) and KEGG pathway analysis to obtain the functional annotations of these co-expressed genes.Gene Set Enrichment Analysis (GSEA) is usually used to determine the statistical significance of a priorly defined set of genes and evaluate the difference between two biological subsets . We usedCD161 expression groups based on the cutoff value of 50%. CIBERSORT was utilized to compare the proportions of 22 tumor infiltration immune cells in both groups. Then, the immune infiltration levels of 24 cell types in BC were downloaded from published literature. ssGSEA analysis was performed to assess the association between CD161 expression and immune infiltration. TISIDB (http://cis.hku.hk/TISIDB/), TIMER and GEPIA were used to verify this association.All 1097 BC patients enrolled from TCGA database were divided into high and low CD161 expression value as a cutoff point, all patients were divided into high CD161 and low CD161 expression groups for survival analysis, GSEA analysis and immune infiltration analysis. The follow-up threshold of OS, DFS and DSS were displayed in the survival curve.Our study was designed according to the reporting guideline checklist Tripod . The RNAt-test was performed to evaluate the difference in CD161 expression between paired tissues. The binary logistic model was performed by R package ISLR. The Cox regression and nomogram analyses of survival were conducted by R package survival, ggforest and RMS. The GO and KEGG analyses were accomplished by R package clusterProfiler. The CIBERSORT R script and R package GSVA were used to evaluate the immune infiltration. All the tests were two-sided, and p < 0.05 was defined as statistically significant.All statistical analyses were conducted using R-4.1.2. In stratification analysis, the case was deleted from the data set when the stratified variable was missing. The paired sample CD161 was lower in the tissue samples of most malignant tumors, like bladder cancer (BLCA), breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD)  . Oncomin samples . This di samples . The cliCD161 expression groups, based on the cutoff value of 50%. The case was deleted from the data set when the stratified variable was missing. The binary logistic model showed that lower CD161 expression was associated with higher T stage , distant metastasis , and higher pathologic stage . Meanwhile, there was no statistical correlation between CD161 expression and other clinicopathological parameters, like lymph node stage, PR status, ER status, and anatomic neoplasm subdivision   , PR status , and age  were independent prognostic factors for OS in BC  . We next= 0.032) . MoreoveOS in BC . Therefo and age . The cal and age .CD161 in BC, we performed GSEA analysis in high and low CD161 expression groups, and GO and KEGG enrichment analyses in CD161 co-expressed genes. In the GSEA analysis, NK cell-mediated cytotoxicity pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, antigen processing and presentation, cell adhesion molecules cams, cytokine-cytokine receptor interaction, primary immunodeficiency, and hematopoietic cell lineage were significantly enriched in CD161 low expression group, according to NES, NOM p values, and FDR values  cells were upregulated in the high CD161 expression group. On the contrary, the proportions of resting dendritic cells, M0 macrophages, neutrophils, activated natural killing (NK) cells, and CD4+ memory activated T cells were upregulated in the low CD161 expression group , cytotoxic cells , B cells   . TIMER dic cells . TISIDB KT cells . We furthaustion . The resKLRB1, is a C-type lectin-related type II transmembrane protein, which belongs to the natural killer cell receptors  implied its outstanding predictive and discriminative power. These results demonstrate that CD161 can serve as a favorable prognostic biomarker for BC. In diffuse glioma and hepatocellular carcinoma, CD161 exerts carcinogenic effects by multiple cancer-related signaling pathways, including CD161-CLEC2D pathway, TCR signaling pathway and cytokine-cytokine receptor interaction (CD161 in BC have not been fully elucidated and deserve further exploration. In our study, the GSEA analysis showed that NK cell-mediated cytotoxicity pathway, TCR signaling pathway, BCR signaling pathway were most significantly enriched in CD161 low expression group. The KEGG analysis showed that CD161 co-expressed genes were enriched in pathways of hematopoietic cell lineage, cell adhesion, and cytokine-cytokine receptor interaction. These findings suggest that CD161 may affect the survival outcomes of BC patients by regulating cancer-related immune response. This theoretical hypothesis requires further experimental validation. TILs are indispensable to an intact immune response to cancer, and their prognostic value has been verified in many solid tumors (CD161 define CD4+ T cells, thus improving the prognosis of OPSCC (CD161 expression and immune infiltration. The results indicated a remarkably positive association between CD161 expression and immune infiltration of B cells, CD8+ T cells, CD4+ T cells, and NK cells. Moreover, CD161 expression was also positively correlated with the levels of immune markers of NK cells, T cell, B cells, and T cell exhaustion, further validating the relevance between CD161 expression and immune infiltration. Based on the results of survival analysis, functional enrichment analysis and immune infiltration analysis, we speculated that CD161 may regulate immune infiltration to inhibit tumor progression and improve prognosis. We should realize several limitations of this study. First, it is a retrospective analysis based on existing public databases, and some important clinical information is not available. Selection bias and missing data may contribute to inaccuracy of results. Besides, the in vitro and in vivo experiments were not carried out to confirm the results. Last, the enrichment analysis was not enough to figure out the specific mechanisms involving CD161-related immune signaling pathways. The mechanism of CD161 in regulating the immune cell infiltration in BC should be further explored.Due to the heterogeneity of breast tumor, the current clinicopathological staging and typing systems that provide clinical decision support and assist survival outcome prediction still have some limitations . New theeceptors . Among Neceptors . Among Teceptors . Previouoma etc. . Althoug results . By Cox eraction . Neverthd tumors . Previouof OPSCC . AccordiCD161 is an independent prognostic factor in BC, and a high expression of CD161 is significantly correlated with favorable clinicopathological parameters, better clinical outcomes and increased immune infiltration. CD161 may serve as a potential prognostic and therapeutic biomarker for BC.


Nymphoides peltata is an aquatic floating weed widely distributed in the northern hemisphere of Eurasia. To better determine its phylogenetic relationships with other Nymphoides species and other aquatic plant species, the chloroplast genome of N. peltata was sequenced. The chloroplast genome size is 152,198 bp, consisting of a large single-copy region  and a small single-copy region  separated by a pair of inverted repeats with a length of 25,079 bp. The chloroplast genome contains 127 genes, including 85 protein-coding genes, 34 tRNA genes, and eight rRNAs. The maximum-likelihood phylogenetic tree showed that N. peltata is more closely related to other Nymphoides species, providing new insight into the evolution and genetic diversity of aquatic weeds. Nymphoides peltata (S.G.Gmel.) Kuntze (Revis. Gen. Pl. 1891) is an aquatic floating weed which belongs to the family Menyanthaceae. N. peltata is widely distributed in temperate and subtropical regions of the northern hemisphere . Collection and research of plant material were carried out by guidelines provided by Hainan University Ethical Committee and comply with local legislation. The voucher specimen (Np-WWF-20210625) was deposited at Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresource . Total genomic DNA was extracted from leaves and sequenced through Illumina NovaSeq 6000 by the Beijing Novogene Bioinformatics Technology Co., Ltd. . The whole chloroplast genome DNA was assembled by the NOVOPlasty v4.2  and a small single-copy region  separated by a pair of inverted repeats (IR) with a length of 25,079 bp. It was annotated with 127 genes, including 85 protein-coding genes, 34 tRNA genes, and eight rRNA genes. The 85 protein-coding genes contain photosynthesis subunit, RNA polymerase, ribosome components, maturase, protease, membrane transporter, and five proteins with unknown functions. The sizes of tRNA were from 77 to 88 bp. The lengths of rRNA were 103 bp (rrn4.5), 121 bp (rrn5), 1490 bp (rrn16), and 2809 bp (rrn23).The complete chloroplast genome of N. peltata, the phylogenetic analyses were performed based on chloroplast nucleotide sequences of six Nymphoides species, and four Nymphaea species (family Nymphaeaceae) were selected as the outer groups. The result supported that N. peltata has the closest phylogenetic relationship with Nymphoides hydrophylla (Nymphoides species formed a cluster. Consistent with previous results (Ho and Ornduff Nymphoides formed a monophyletic clade in aquatic weeds. The complete chloroplast genome of N. peltata provides valuable genetic information for improving our understanding of the evolution of aquatic weeds, exploring genetic variations, and designing conservation strategies.To investigate the evolutionary relationship of rophylla . MoreoveWW designed the research. DW, ZW, and YZ collected the specimens. XZ, YL, and JZ performed the experiments and analyzed the data. All authors approved its final version.


Child abuse is associated with a wide range of mental disease including psychotic disorders. Few studies have investigated the role of child abuse in contributing to increase the risk of psychosis in migrant population.To explore the risk of first episode psychosis (FEP) in migrants and natives for each type of trauma i.e. physical abuse (P.A.), sexual abuse (S.A.), emotional abuse (E.A.), physical neglect (P.N.) and emotional neglect (E.N.).Within a large case- control incidence sample of FEP from the EU-GEI study  we evalued the assocition of childhood trauma with FEP in migrants and natives. Associations were adjusted for age, gender, social status, level of education, family history of psychosis and cannabis use. Trauma was assessed through Childhood Trauma Questionnaire (CTQ).CTQ mean score was higher in FEP migrants  than in FEP natives  (p = 0.002). In natives every type of child abuse was associated with FEP. In migrants P.A., S.A., P.N. were associated with FEP. We found a dose – dependent relationship between trauma and FEP.Child abuse is common in individuals with psychosis. FEP migrants are more exposed to childhood trauma. Clinicians should routinely assess patients for childhood trauma. When treating a FEP migrant patient, clinicians must be aware of an underlying traumatic childhood adversity more than of a traumatic migration history.


Mobile genetic elements (MGEs) mediate the shuffling of genes among organisms. They contribute to the spread of virulence and antibiotic resistance (AMR) genes in human pathogens, such as the particularly problematic group of ESKAPE pathogens. Here, we performed the first systematic analysis of MGEs, including plasmids, prophages, and integrative and conjugative/mobilizable elements (ICEs/IMEs), across all ESKAPE pathogens. We found that different MGE types are asymmetrically distributed across these pathogens, and that most horizontal gene transfer (HGT) events are restricted by phylum or genus. We show that the MGEs proteome is involved in diverse functional processes and distinguish widespread proteins within the ESKAPE context. Moreover, anti-CRISPRs and AMR genes are overrepresented in the ESKAPE mobilome. Our results also underscore species-specific trends shaping the number of MGEs, AMR, and virulence genes across pairs of conspecific ESKAPE genomes with and without CRISPR-Cas systems. Finally, we observed that CRISPR spacers found on prophages, ICEs/IMEs, and plasmids have different targeting biases: while plasmid and prophage CRISPRs almost exclusively target other plasmids and prophages, respectively, ICEs/IMEs CRISPRs preferentially target prophages. Overall, our study highlights the general importance of the ESKAPE mobilome in contributing to the spread of AMR and mediating conflict among MGEs. Mobile genetic elements (MGEs) are DNA entities that are capable of capturing and shuffling genes intra- and intercellularly . CoevoluMGEs carry non-essential genes that can provide their bacterial host with adaptive functions and alter their fitness, such as antimicrobial resistance (AMR) and virulence genes ,19. ThesEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa) and one genus (Enterobacter sp.)  ,32. Thester sp.) . The WHOter sp.) . AMR andter sp.) ,33, and ter sp.) ,41. Receter sp.) .In this study, we performed the first systematic analysis of the ESKAPE pathogens mobilome. We asked (i) how prevalent are different MGEs  across the ESKAPE pathogens; (ii) how broad or constrained is the combined MGEs’ network; (iii) which functions are overrepresented in these MGEs, and if AMR and virulence genes are differently distributed in pairs of conspecific ESKAPE pathogens with and without CRISPR-Cas systems, which we here focus on as examples of effective defense systems in bacteria ,25; (iv)https://github.com/kblin/ncbi-genome-download). Genomes listed as ‘unverified’ were removed from our dataset. We also excluded genomes classified as ‘Enterobacteriaeceae’. Finally, we used pyANI v0.2.10 (https://github.com/widdowquinn/pyani) to calculate the average nucleotide identity based on MUMmer (ANIm) and removed genomes with an ANIm value below the 95% threshold for species delineation  . Multi-locus sequence typing (MLST) profiles were determined with mlst v2.19.0 (https://github.com/tseemann/mlst). The curated genomes were automatically annotated using Prokka v1.14.6 (https://github.com/tseemann/prokka) , using ncbi-genome-download v0.3.0   and the GToTree) . We used/prokka) .Since the ESKAPE pathogens (as most bacteria) are haploid, we separated the large replicon (i.e. the chromosome) from the extrachromosomal replicons. For the latter, only accessions with ‘plasmid’ and ‘complete sequence’ on their description were kept and were used for further plasmid analyses.https://github.com/labgem/PPanGGOLiN) (https://bedtools.readthedocs.io/en/latest/) (http://hmmer.org/)  , which unGGOLiN) . This melatest/)  to extraer.org/)  against er.org/) . Simultahttps://ftp.ncbi.nlm.nih.gov/refseq/release/viral/, accessed on the 25 January 2022). The masked chromosomal genbank files were then used as input in phispy v4.2.6 , which combines similarity- and composition-based strategies to look for prophages . We used the Analyzer function in Cytoscape to compute a comprehensive set of topological parameters, such as the clustering coefficient, the network density, the centralization, and the heterogeneity.To estimate the pairwise distances between all ESKAPE MGE types , we first ran the MMSEQseqs2 v13.45111 package , using 9/bindash). Each MGhttps://www.ncbi.nlm.nih.gov/research/cog-project). The alignments were performed with DIAMOND v0.9.10.111 (https://github.com/kkpenn/merger_COG2020/blob/main/merger_2.py) and DIAMOND makedb with default settings. Around 36, 38 and 55% of the proteins encoded in plasmids, ICEs/IMEs, and prophages matched a protein in the COGs database, respectively, and were therefore annotated with the information of their corresponding homolog. Lists of non-redundant COG definitions (e.g. COG0105) were extracted separately for prophages, plasmids and ICEs/ IMEs, and compared with venny v2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to identify unique and shared COGs. Likewise, COGs occurrence was determined separately for the proteomes of the three MGE types in the different ESKAPE. Information on the COGs classification into functional categories was retrieved from https://ftp.ncbi.nih.gov/pub/COG/COG2020/data/. The relative frequency of the different COG functional categories per MGE/ESKAPE pair was calculated by summing up the occurrences of COGs belonging to a given functional category and dividing the resulting number by the total number of proteins observed in the corresponding MGE/ESKAPE pair.COGs annotation of the MGE proteins was carried out through sequence alignments against the COGs 2020 database  and clustered them using the cluster algorithm from the MMseqs2 package . The prohttps://github.com/tseemann/abricate) to scan extracted MGEs and masked genomes against antimicrobial resistance and virulence genes (using pre-downloaded databases from Resfinder (https://github.com/Russel88/CRISPRCasTyper), including a maximum of 3 unknown genes between Cas genes in the operon, an overall E-value threshold of 0.01, and a 10kb distance threshold to connect Cas operons and CRISPR arrays (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs), when using MGE CRISPR spacers as a query. CRISPR spacers from masked genomes were also blasted against a local database of our extracted (non-masked) MGEs. Hits with at least 95% nucleotide identity and 95% sequence coverage were considered as spacer targets , a substantially smaller collection of ICEs/IMEs is available at ICEberg database (n = 1325), and was last updated in September 2018. Due to this limitation in the number of publicly available ICEs/IMEs sequences, and to have a representative collection of these three different types of MGEs, we focused on the curated dataset presented in this study to look for targets of CRISPR spacers. Additionally, we mapped the CRISPR spacers against annotated genes across the ESKAPE mobilome, using the same blast approach and the same thresholds.We used abricate v1.0.1 . This collection was used to build a local database with DIAMOND (https://github.com/bbuchfink/diamond) (We retrieved an Anti-CRISPR collection of 1111 non-redundant proteins from Anti-CRISPRdb v2.2 (diamond) . We useddiamond) , currentP-values adjusted using the Holm–Bonferroni method. Comparisons between pairs of conspecific genomes with and without CRISPR-Cas systems, as well as between MGE targets for CRISPR spacers, were performed using the Wilcoxon test, and the p-values adjusted using the Holm–Bonferroni method. Values above 0.05 were considered as non-significant (ns). We used the following convention for symbols indicating statistical significance: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.Comparisons between MGEs’ GC content and sequence length were performed using the Kruskal–Wallis test, and the Enterobacter sp. genomes and type strains belonging to the Enterobacteriaceae family  were removed for each ESKAPE species . Since te family . Our cure family . We foune family  and B. Te family . S. aure Figures  and C. T Figures . Curiouss Figure .E. faecium and P. aeruginosa (∼3 elements/genome)  Figure  and C. A) Figure .K. pneumoniae (340/717). Our results show that different MGEs are asymmetrically distributed across the ESKAPE pathogens, with K. pneumoniae genomes taking the lead for the co-occurrence of ICEs/IMEs, plasmids and prophages.When looking into the presence/absence combination of co-occurring MGEs across the ESKAPE pathogens, we noticed that the most frequent combination involved the presence of the three MGEs . With the exception of S. aureus, we observed that plasmids across the ESKAPE pathogens show more variation in size when compared with ICEs/IMEs and prophages , and a weak negative correlation between the prophages’ GC content and sequence length  was used as a measure of DNA sequence similarity between all MGE pairs. The majority of MGE pairs shared little similarity, with a JI value below 0.25 , and S. aureus and E. faecium in the second cluster. MGEs within these promiscuous clusters tend to be more dissimilar than those assigned to densely connected and ESKAPE/MGE pair-restricted clusters. We observed high genetic similarity between different MGE/ESKAPE pairs  encoded by MGEs identified in this study. COGs are protein sets conserved across lineages that typically share function and are therefore used for systematic function prediction in poorly characterised genomes . COGs haE. faecium than in other ESKAPE . Interestingly, we detected some variation in the relative contribution of MGE proteomes to different COG functional categories among the ESKAPE pathogens . We observed that AMR genes are broadly distributed in plasmids across the ESKAPE pathogens. Even though the total number of prophages far outnumber that of plasmids in our collection  were prevalent among proteobacterial representatives of the ESKAPE pathogens but were mostly absent from S. aureus and E. faecium , chloramphenicol (cat genes), trimethoprim (dfr genes) and tetracyclines (tet genes) were found in all representatives of the ESKAPE pathogens. Genes involved in resistance to vancomycin (the vanHAX and vanHBX gene clusters) were exclusively found in S. aureus and E. faecium, while genes coding resistance for quinolones (qnr genes) and colistin (mcr genes) were only found in the proteobacterial representatives , and those that promote target site modification . These virulence loci were mostly present in ICEs/IMEs, as previously reported , when compared with masked genomes  consisted entirely of either CRISPR-Cas positive or negative genomes (A. baumannii from our dataset was ST2 (n = 101), which only included strains with no CRISPR-Cas systems. On the other hand, the second most prevalent MLST profile in this species , only consisted of strains with I-F CRISPR-Cas systems. The most frequently observed MLST profile from K. pneumoniae , consists mostly of CRISPR-Cas negative strains . The four strains with positive hits carried IV-A3 CRISPR-Cas systems on plasmids. ST258 (n = 47) was the second most common K. pneumoniae MLST profile identified in our dataset, and again, consisted entirely of strains with no CRISPR-Cas systems. However, ST147 and ST15  carried I-E CRISPR-Cas systems in all strains. Finally, looking at P. aeruginosa, we found that the most prevalent MLST profiles in our dataset  carried no CRISPR-Cas systems. The only exception was found in a ST235 strain, which carried an I-C CRISPR-Cas system on an ICE/IME. In contrast, ST233 and ST1971 (n = 8 for both) consisted exclusively of strains carrying the I-F CRISPR-Cas system on masked genomes , ICEs/IMEs (n = 7 I-C in P. aeruginosa and n = 1 III-A in S. aureus), and prophages   Figure . The pla) Figure , which rP. aeruginosa, for which CRISPR-Cas positive genomes were significantly smaller than their counterparts , as observed before . The non-significant differences observed for A. baumannii, Enterobacter sp. and S. aureus could in part be explained by the low sample size of CRISPR-Cas positive genomes . Curiously, we noticed that CRISPR-Cas positive genomes in K. pneumoniae were GC poorer . Given the known association between GC content and genome size , in accordance to a previous study . Given that P. aeruginosa genomes positive for these systems are significantly smaller than their CRISPR-Cas negative counterparts , confirming our prediction that the genome size was a confounding variable obscuring the effect of CRISPR-Cas systems on the prevalence of virulence genes in P. aeruginosa.Since virulence genes are overrepresented in the chromosome Figure , we asseterparts , the lowK. pneumoniae . Indeed, AMR genes were more prevalent on ICEs/IMEs from P. aeruginosa genomes with CRISPR-Cas systems . Taken together, we observed species-specific trends shaping the number of MGEs, AMR and virulence genes across pairs of conspecific ESKAPE genomes with and without CRISPR-Cas systems.We then explored whether CRISPR-Cas presence or absence reduced the number of MGEs acquired in pairs of conspecific ESKAPE pathogens. We would expect to detect a smaller number of MGEs in genomes harbouring these immune systems. However, this trend was only observed in e Figure . The vare Figure . Finallye Figure . For mos systems . A simil systems . CuriousCEs/IMEs , was recCEs/IMEs . Regardin = 410) antagonising CRISPR-Cas subtypes I-E, I-F and II-A were identified across prophages, ICEs/IMEs and prophages from all ESKAPE pathogens except S. aureus , we used this collection as a database and took the CRISPR spacers identified in masked genomes as a query. In parallel, we used the CRISPR spacers identified in MGEs as a query and the MGEs collection as a database. For the latter, and to avoid self-targeting hits, we masked all CRISPR spacers from the MGEs collection used as database. We observed that only a small fraction of MGEs carry CRISPR spacers . A total of 1087 spacers was found across all MGEs . Given the large number of MGEs and CRISPR-Cas-encoding plasmids in K. pneumoniae . Across the S. aureus genomes, we found a total of 174 CRISPR spacers  and only four Cas operons, which explains the low prevalence of CRISPR-Cas systems in this species  of CRISPR spacers in ESKAPE masked genomes yielded matches to spacer targets in MGE sequences, found on 16.4% MGEs . Studying MGEs in parallel allowed us to explore their uneven distribution across a collection of complete genomes from important pathogens, and to explore potential DNA sharing events between different MGE types. By separating these elements from masked (MGE-free) genomes, we were able to observe an overrepresentation of AMR genes and anti-CRISPRs across the ESKAPE mobilome. Furthermore, we focused on CRISPR-Cas as an example of bacterial defense systems, which are efficient against invading DNA and provide information on their targets through analysis of the associated spacer sequences , and which include representatives of both Proteobacteria and Firmicutes, and also phylogenetically divergent bacteria , or intTo conclude, our results indicate that prophages, ICEs/IMEs, and plasmids are asymmetrically distributed across the ESKAPE pathogens. We found that these MGEs can be found in multiple genera (and phyla), even though most clusters are constrained by host similarity and/or the type of MGE. We observed that the proteome of ESKAPE MGEs is highly diverse, involved in diverse functional categories, and features convoluted distribution patterns (including both MGE/ESKAPE specific and widespread proteins). When comparing against masked (MGE-free) genomes, we observed the pervasive association of AMR genes and anti-CRISPRs with the ESKAPE mobilome. We also found different targeting biases to CRISPR spacers found on plasmids, prophages, and ICEs/IMEs, highlighting their genetic independence. Taken together, our results illustrate the potential of network-based approaches and comparative genomics to underscore the composition and dynamics of gene flow across different MGEs, and sheds a new light in the role of the overlooked ICEs/IMEs as important players in the MGE-MGE warfare in the ESKAPE pathogens and thus the main groups of highly problematic human pathogens.https://gitlab.gwdg.de/botelho/eskape_paper).Analyses were made with a combination of shell and R 4.0.3 scripting. R scripts and supporting tables used to create the figures are available at gkac1220_Supplemental_FilesClick here for additional data file.


Bipolaris sorokiniana (teleomorph Cochliobolus sativus) is one of the devastating diseases of wheat in the warm and humid growing areas around the world. B. sorokiniana can infect leaves, stem, roots, rachis and seeds, and is able to produce toxins like helminthosporol and sorokinianin. No wheat variety is immune to SB; hence, an integrated disease management strategy is indispensable in disease prone areas. A range of fungicides, especially the triazole group, have shown good effects in reducing the disease, and crop-rotation, tillage and early sowing are among the favorable cultural management methods. Resistance is mostly quantitative, being governed by QTLs with minor effects, mapped on all the wheat chromosomes. Only four QTLs with major effects have been designated as Sb1 through Sb4. Despite, marker assisted breeding for SB resistance in wheat is scarce. Better understanding of wheat genome assemblies, functional genomics and cloning of resistance genes will further accelerate breeding for SB resistance in wheat.Spot blotch (SB) caused by  Bipolaris sorokiniana (teleomorph Cochliobolus sativus) syn. Drechslera sorokiniana, syn. Helminthosporium sativum is the most devastating disease of wheat grown in warm and humid areas. In Eastern Gangetic Plains (EGP) of India, Bangladesh and Nepal, B. sorokiniana appears in a complex with Pyrenophora tritici-repentis (Died.) Drechs. (anamorph Drechslera tritici-repentis (Died.) Shoemaker) responsible for tan spot (TS) and is commonly known as Helminthosporium leaf blight (HLB) (Spot blotch (SB) caused by the hemibiotrophic fungus ht (HLB) . Occurreht (HLB)   . Under fht (HLB) , with anht (HLB) .B. sorokiniana causes a typical symptom of light brown colored lesions of oval or oblong to elliptical shape on leaves, sheath, nodes or glume of its host plants. Size of the lesion gradually increases, then partial to whole leaf may become chlorotic, turning brown and drying up  and genomic selection. The present work aims to summarize the most important findings, especially those reported in recent years, in the area of SB management in wheat.in wheat . No posiin wheat . Cross iin wheat . A numbeB. sorokiniana isolates is sufficiently high  types. Under controlled conditions, sexual spores of B. sorokiniana were isolated from barley (The pathogen produces olive brown mycelia and light grayish colonies on potato dextrose agar (PDA) medium at early stage, which turns into black at later stage. Conidia are brown colored, elliptical, straight or curved multiple celled with 3-9 septa tapering at the ends, measuring 10-28 × 40-120 µm . Variabitly high . Bipolarm barley  and recem barley .B. sorokiniana, which damages membrane permeability and affects mitochondrial oxidative phosphorylation and chloroplast photophosphorylation  marker SCARBSokiniana . Alternain China . Using u markers .B. sorokiniana from India, Australia, USA and China are currently available (https://www.ncbi.nlm.nih.gov/data-hub/genome/?taxon=45130). A highly virulent isolate BS_112 (GenBank accession number KU201275) from India has a genome size of 35.64 Mb and 10,460 genes were predicted with an average gene length of 435-545 bp and gene density of 250-300 genes/Mb  loosens the cell wall by cleaving α-(1→4) linkages of the homogalacturonan, an important constituent of the middle lamella of the cell wall  for SB resistance varied greatly in different studies, e.g., from 0.21 to 0.64 in Quantitative nature of SB resistance is predominantly reported in wheat , but repSb1 through Sb4. Sb1 was mapped on chromosome arm 7DS, being co-located with Lr34  (tsn1 (Genome wide association studies (GWAS) have been widely performed to detect SB resistance QTLs in wheat. In a study using 566 spring wheat landraces, 1 (Lr34)  and tsn14)  consistent over the locations , comprising high yielding SB resistant genotypes for testing over the different countries in SA, Africa and Latin America . Testingocations .via transferring Qsb.bhu-2A and Qsb.bhu-5B from Chirya 3 and Qsb.bhu-2A from Ning 8201 into the genetic background of HUW 234 (The progress in achieving genetic gain for SB resistance is slow, due to reasons like quantitative inheritance, moderate heritability, strong genotype × environmental interaction, and high variability of the pathogen over time. Phenotypic selection is often associated with confounding traits, and molecular markers can be used to assist the pyramiding of resistance QTLs and selection for SB resistant genotypes . Superio HUW 234 . Increas HUW 234 . HoweverGenomic selection can improve the efficiency of breeding program by reducing phenotyping cost, time and increasing selection intensity and genetic gain. Studies on genomic selection for SB in wheat are limited, and a successful example was reported by B. sorokiniana was reported by Gene silencing through RNAi is a powerful tool for controlling insects, nematodes, viruses, fungal diseases like powdery mildew, and rusts . UtilizaPvPGIP2 (TaPIMP1 and TaPIMP2 (B. sorokiniana. However, government regulations on transgenic development are major concerns for researchers.Transgenic lines expressing foreign genes proved to be a potential approach to control insect and diseases in several crop plants. Examples include the heteroexpression of PvPGIP2  and over TaPIMP2  in transAdditional techniques like genome editing  and Eco-Spot blotch is a disease of concern in warmer wheat growing areas of South Asia, Latin America and Africa. Most of the commercially grown cultivars are moderately resistant to susceptible and are subjected to significant yield losses under conducive climatic conditions. An integrated disease management strategy involving cultural practices, chemical control, resistant cultivars, etc., is needed to combat the disease. In addition, modern biotechnology brings new tools for the rapid and efficient development of resistant cultivars in wheat.CR, XH, and PS conceptualized the manuscript. CR drafted the first version. NG and SM collected information and improved the first draft. XH and PS edited and approved the submitted version. All authors contributed to the article and approved the submitted version.


Haemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical.H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65℃, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection.In this study, we used a combined method to detect H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.Taken together, LAMP-LFB could be used as an effective diagnostic approach for The online version contains supplementary material available at 10.1186/s12866-022-02547-5. Haemophilus influenzae is a common human pathogenic strain, which is related to a variety of serious childhood illnesses, such as pneumonia, otitis media, bacteremia and meningitis [H. influenzae from Haemophilus spp., such as Haemophilus parainfluenzae. The traditional detection techniques for isolation and identification of H. influenzae, involving colonial morphology, serological identification and growth assays, are usually consuming a lot of time and cumbersome [ningitis . Previoumbersome . TherefoP6, capsulation-associated protein Bex A [H. influenzae detection. Thereinto, OMP P6 is a highly conserved gene in H. influenzae [H. influenzae [H. influenzae identification than other genes.For clinical detection and analysis, the molecular detection technology has the value of sensitivity and specificity –5. With in Bex A , 8, the fluenzae , 10. Morfluenzae . Hence, Bst DNA polymerase, an appropriate temperature range from 60℃ to 67℃ to make the chain replacement DNA synthesis self-cycle continuously, so as to achieve rapid amplification. After the reaction, the amplification can be judged by the turbidity of the precipitation of magnesium pyrophosphate, the by-product of the amplification, or the fluorescent dye. In this reaction, dumbbell-shaped template was formed first, and then cyclic amplification was carried out, followed by elongation and cyclic amplification [H. influenzae, H. influenzae type b (Hib), and serotype of non-Hib have been detection with LAMP method to diagnose for patients [P6 gene to specifically detect H. influenzae. With strain pure cultures and clinical samples, the optimal reaction conditions, sensitivity, specificity and feasibility of H. influenzae detection strategy were validated.A detection technique of nucleic acid called loop-mediated isothermal amplification (LAMP) was established in 2000 . The prification –14. For fication , 16. In fication , 16. Manfication , 18. Forpatients , 20. Morpatients . In briepatients –25. It ipatients . For theH. influenzae LAMP primers, the LAMP reactions were conducted with H. influenzae, H. parainfluenzae, H. haemolyticus, and non-H. influenzae genomic templates at 63℃ for 1 h. After adding the VDR reagents into the amplification mixtures, the color of positive amplification products of LAMP in tube changed from colourless to light blue , which were used as the templates to analyze the sensitivity of LAMP-LFB assay. As was shown in Fig. H. influenzae by real-time turbidimeter  are all detection methods for LAMP amplification products. Each of those methods has its own advantages and disadvantages. Real-time turbidity method can monitor the reaction in real time, but it needs to rely on turbidimeter. Gel electropheresis, which requires an additional gel electrophoresis process of approximately 30 min. VDR method, although the naked eye can directly identify the reaction results, some negative reaction products also show a slight blue color, which may affect the judgment of the results. Herein, the LFB was employed to analyze LAMP products in P6 gene of H. influenzae was successfully developed. The assay showed high selectivity for H. influenzae detection, high sensitivity of 100 fg in per reaction with pure culture. Meanwhile, the protocol is much more convenient with less time-spending and no expensive equipment. H. influenzae-LAMP-LFB assay established in this study might be used as a diagnosis tool for target pathogens, which would allow clinicians to make better informed decisions regarding patient treatment without delay.The LAMP-LFB assay targeted the specific OMP Bst DNA Polymerase large Fragment was purchased from New England Biolabs Co., Ltd. . The crimson red dye streptavidin-coated polymer nanoparticles  were purchased from Bangs Laboratories, Inc. . The rabbit anti-fluorescein antibody and the biotinylated bovine serum albumin were purchased from Abcam. Co., Ltd. . The FastPure® Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit was purchased from Vazyme biotech co., Ltd . The LA-320C realtime turbidimeter was purchased from Eiken Chemical Co., Ltd . The LA-320C realtime turbidimeter belongs to the special gene amplification assay device for LAMP method. The device can complete the entire process from gene amplification to detection. After incubation under isothermal conditions (60–65℃), the presence of target genes was determined by measuring the turbidity of magnesium pyrophosphate, a by-product of the amplified genes. The turbidity of each sample is measured in real time and the results are graphically displayed on the computer. The turbidity of each sample was measured every 6 s. The measurement is transferred to a computer to confirm the extent of amplification.The Loopamp kits and visual detection reagent (VDR) , 25 wereH. influenzae OMP P6 gene (Genbank accession no. L42023). The specific LAMP primers listed in Table According to the H. influenzae strains, 3 H. parainfluenzae, 3 Haemophilus haemolyticus, 3 Haemophilus parahaemolyticus and 20 non-H. influenzae strains , the sensitivity of LAMP-LFB assays was proceeded with the gradient genomic DNA extracted from pure culture of The effect of LAMP amplification were examined at different times, and the reaction time was set from 10 to 60 min with 10 min interval, which conducted three times. The LAMP product was detected with colorimetric indicators and LFB.H. influenzae, 3 H. parainfluenzae, 3 H. haemolyticus, 3 H. parahaemolyticus and 20 non-H. influenzae strains  from 10 H. influenzae isolates were successfully detected from 22 sputum samples. With DNA Isolation Kit as previously described in Reagents and Instruments part, the genomic DNA were extracted from 22 sputum samples that were positive for H. influenzae and another 33 randomly selected sputum samples that were negative for H. influenzae. The PCR carried out according to Torigoe [H. influenzae in all of the 55 DNA temples. The colorimetric indicators, agarose gel electrophoresis and LFB analysis were used to analyze the LAMP amplification. Then the LAMP-LFB results and PCR results were compared to traditional culture.The 55 sputum samples mentioned in Bacterial Strains and Genomic DNA Preparation part were detected according to traditional culture methods, biochemical identification, colony morphology and Gram stain. As a result,  Torigoe  and LAMPAdditional file 1.