Of the 749 cases, 16.5% were notified in another municipality that was not the patient’s residence. Regarding diagnostic methods, a positive bacterioscopy was adopted for 56% of the patients; culture was not performed for 82% of cases. Histopathological examination was not performed in 90% of the individuals. Rapid molecular test (RMT) was performed in only six (5%) cases. The region needs greater attention focused on diagnostic tests, suggesting that the introduction of RMT and culture by Ogawa-Kudoh could improve the region’s health network to minimise patient displacement and thus avoid the increase in the transmission chain of TB.Tuberculosis (TB) is still considered a priority due to its high incidence rate in Brazil. In this context, we aimed to evaluate the flow of care between the municipalities of patients diagnosed with TB through notification forms of the Information System for Diseases and Notifications (SINAN) in a neglected region of Northern Brazil, Ilha do Marajó, state of Para. For this, we performed a descriptive, retrospective study on data obtained from the National Register of Health Establishments and SINAN from 2013 to 2018. We used Pearson’s Chi-square and G Test with  Mycobacterium tuberculosis complex, remains a major cause of morbidity and mortality worldwide. Brazil is among the 30 countries with the highest TB burden globally [Tuberculosis (TB), caused by the globally . Examiniglobally .Programa Nacional de Controle da Tuberculose”—PNCT) was initiated to control TB in Brazil, including the creation of Primary Health Care (PHC) to improve access to diagnosis, treatment, prevention, and surveillance for all users of the Brazilian Unified System of Health (“Sistema Unico de Saúde”—SUS). In this context, PHC has a fundamental role in the programme, as the Health Care Network (HCN) is the gateway for patients. However, health services are still conducted in a fragmented and episodic manner [The National TB Control Programme  in Brazil [Sputum smear microscopy (bacterioscopy) and rapid molecular tests (RMT) are the main methods employed in detecting cases and early diagnosis of TB . Howevern Brazil .Therefore, considering the need to enhance the healthcare network (HCN) in this region, this study aimed to comprehensively describe the epidemiological profile of TB cases in Ilha Marajó. For this, we analysed the patients’ municipality of residence and the municipality where TB cases were reported. The primary focus was evaluating the flow of attention to TB cases in Ilha do Marajó using data obtained from the Brazilian Notifiable Diseases Information System (SINAN) in the state of Pará.We conducted a descriptive, retrospective study that proposed to analyse the flow of TB patients in healthcare units to analyse and describe the inter-organisational structure characteristics of the health system and the networks of social interactions related to patients from TB in Ilha do Marajó.2 in extension, is bathed to the northwest by the mouth of the Amazon River and is separated from the mainland to the south by the Pará River, which, to the southeast, expands receiving the waters of the Tocantins River and other smaller rivers, then changing its name to Baía do Marajó [2 [The study population consisted of all new TB cases notified to the SINAN from 2013 to 2018. The study was conducted at the Ilha do Marajó, the largest island in this archipelago; it has 104,139.93 kmo Marajó . Its totThe study encompassed all newly reported TB cases occurring in Ilha do Marajó between 2013 and 2018. Cases lacking a complete address in the SINAN database were excluded from the study.Secretaria de Saúde Pública do Estado do Pará”—SESPA). The number of TB cases in Ilha do Marajó was 749; 17% (124) of cases were reported in a municipality other than that of residence. According to the Brazilian National Health System Information Technology Department (DATASUS). The Ilha do Marajó has 16 municipalities, shown in For data collection, we used the database made available by the Pará State Department of Public Health  using ArcGIS 10.4 software  . We obtained the state, municipal, micro and mesoregion limits from the Brazilian Institute of Geography and Statistics (IBGE) [The municipality of residence and notification were geocoded with Google Maps and OpenStreetMap (s (IBGE) .In the patient flow map, the colours of the lines represent the flow intensity between the municipality, which is the number of patients notified in another municipality. We established four thorough quartiles: light green , dark green (medium flow: two to four cases), yellow (intermediate flow: five to eight cases), and red (intense flow: nine to fourteen cases).Notification of immigrant cases and the number of cases of residents notified by health establishments  were geocoded, divided into quartiles: 1 to 7 cases; 8 to 40; 41 to 85 cases; above 85 cases.www.cnes.datasus.gov.br, accessed on 20 February 2023), and only the notifying units were geocoded by Google Maps using ArcGIS 10.4 software. We considered primary care establishments  as well as medium/high care . We used Kernel’s estimate for patients who sought other healthcare facilities. Thus, it was possible to analyse the behaviour of the point patterns and provide the point intensity of the process throughout the studied area through Interpolation. It was applied using the parameters of the quartic function and 1.5 km radius, divided between absent (dark green), low (light green), medium (yellow), high (orange), and very high (red).We extracted the information on health establishments from the National Register of Health Establishments (CNES) . For the tabulation of data, we used the statistical programme Statistical Package for the Social Science (SPSS) and EpiInfo v7.2 in a Windows 7 environment. For the descriptive analysis, we performed Pearson’s Chi-square test (Wilks’ G2) for independence, adopting a significance level of The study was approved by the Research Ethics Committee of the Universidade do Estado Pará (UEPA) under the number 3,705,199.From SINAN, we collected 124 cases which referred to locations beyond the patient’s municipality of residence. Among these cases, 73% (90/124) were reported in the metropolitan region of Belém, which includes Belém, Marituba, and Ananindeua. Additionally, 2% (3/124) were reported in the northeast region of Pará, covering Abaetetuba, Barcarena, and Limoeiro do Ajuru. The remaining 25% (31/124) were reported in various municipalities within Marajó.p-value < 0.05). Upon comparing the two sample groups, it is evident that there is no statistical difference in sociodemographic variables.Regarding the clinical characteristics, 78% (97/124) were pulmonary TB . Among tDespite the notified cases, 25 (20%) patients did not undergo diagnostic bacterioscopy, and 30 (24%) reported a negative result. Of the patients with negative bacterioscopy (30), only twelve (40%) underwent culture, four (13%) were positive, two (7%) were negative, and six (20%) were being analysed at the time. Of the 124 cases, 23 (18%) underwent culture, 6 (5%) underwent RMT for TB, 2 (2%) of which were susceptible to rifampicin, and 2 (2%) were inconclusive.When examining the association with directly observed treatment (DOT), 16 cases (31%) were recorded as following this treatment approach, while 51 cases (41%) did not adhere to this modality, and 57 cases (46%) were not even registered in the notification form. Regarding the outcome, more than half of the cases achieved cure (55%). In comparison, there were 9 cases (7%) of treatment dropout, 4 cases (3%) resulting in death due to TB, 1 case (1%) of drug-resistant TB, and 36 cases (29%) were transferred to their respective municipality’s health unit for continued treatment after diagnosis.The most significant patient flow is directed towards the state capital, Belém, primarily originating from the municipalities of Soure , Salvaterra , Ponta de Pedras , and São Sebastião da Boa Vista . These municipalities are in closer geographical proximity to the capital. Another noticeable flow is from Bagre to Breves, where nine cases (58.3%) of Bagre residents were reported in Breves. Additionally, there are intermediate flows, such as from Santa Cruz do Arari to Belém , Portel to Belém , and Melgaço to Breves .Ilha do Marajó comprises 238 health facilities, out of which 67 facilities (28%) were notified as active, and 3 were inactive, specifically located in the municipality of Breves. However, even during their inactive status, there were cases reported for the period they were active. These health establishments were categorised into two groups: Basic Care facilities, including ambulatory, health centres, health posts, and mobile river units, which accounted for 223 units (93%), and Medium/High complexity facilities, including mixed units and hospitals, which represented 18 establishments (7%). Among these establishments, 8 operate under dual management , 1 is under state management, and the majority, 232 facilities, are solely under municipal control .The municipality with the highest number of health facilities was Breves, with 36 active and 3 inactive units, but only 5 units reported cases. Within the island, this municipality receives the most patients from other municipalities. The municipality with the lowest number was Santa Cruz do Arari, having six establishments (one notified), with 22% of its residents reported in Belém.Of the municipalities outside the Ilha do Marajó, the capital Belém had 19 establishments notifying residents of Marajó, with 11 (58%) of primary care and eight (42%) of medium/high complexity, making it the hospital of reference for infectious and contagious diseases that reported the most cases  and the second was a unit specialised in particular infectious and parasitic diseases .Exploring social networks in the health sciences field originated from comprehending the dynamics of microorganism transmission within confined spaces, specifically through the interactions between their components (patients) and their movements in the area. According to Nilo et al. , this anThe clinical and epidemiological characteristics observed in this study resemble those identified in a previous study by Mesquita et al.  on TB caMycobacterium tuberculosis. One of the critical foundations of the PNCT is the decentralisation of TB control actions to PHC units. These units are deemed the focal point for managing all prevention and control measures, ensuring comprehensive and continuous care. These measures include actively searching for suspected cases displaying symptoms, conducting diagnostic investigations, monitoring confirmed cases, supervising treatment, and performing regular bacterioscopy for control [TB continues to possess stigmatising characteristics, shedding light on the precarious social conditions, including living conditions , experie control .In this study, only 16 patients 13%) entered the DOT mode, and in the state of Pará in 2022, 25.1% of patients in were DOT % enteredFollowing the guidelines set by the World Health Organization (WHO) for tuberculosis (TB) control, it is recommended to attain a cure rate of 85% or higher while keeping the abandonment rate below 5% . RegrettIlha do Marajó is renowned for its extractive production (fruit and fisheries) and livestock farming. However, it is crucial to acknowledge that this region is characterised by extreme poverty, with municipalities exhibiting the lowest HDI in Brazil ,17. WhenThe transmission dynamics of tuberculosis (TB) are influenced by the shift within the region due to its aerosol-transmissible nature. This area’s central displacement mode relies on river transport, with rivers and boats serving as the primary means of transportation for daily activities. However, river transport in Pará is often overcrowded, exceeding seating capacity and resulting in extended travel times between municipalities. This crowded and prolonged travel environment significantly facilitates the spread of infectious diseases.The combination of unstable social policies and limited healthcare services in Ilha do Marajó leads TB patients to seek primary healthcare in neighbouring municipalities, creating spatial heterogeneity. This spatial heterogeneity refers to the transmission of infectious diseases related to travel. Apart from intensifying the transmission chain, the dispersion of infected individuals hampers disease control compared to cases that are not dispersed ,18,19.In Ilha do Marajó, we observed patient movement patterns towards the municipality of Breves, which hosts many healthcare facilities, including those offering medium/high complexity services close to the state capital, Belém. Regarding Belém, we noticed a higher concentration of TB cases in specialised sites for infectious diseases, which raises the likelihood of transmission chains during this displacement, mainly due to long sea journeys where individuals are confined on boats for hours until reaching their destination.According to Azevedo et al. , patientIt is worth emphasising that a strategy capable of effectively reducing the number of patients seeking emergency care involves conducting home visits and providing guidance to community health agents. Assume these agents become aware of or identify someone experiencing a cough lasting three weeks or longer, in this case, referring that individual to a healthcare facility for necessary examinations is essential. Additionally, fostering a stronger connection between the health service, healthcare professionals, individuals, families, and the community plays a crucial role in this approach .Understanding the organisation of PHC and the search for care in TB transmission is a central factor in controlling diseases in risky places. This study, conducted in Ilha do Marajó, revealed a noteworthy pattern wherein patients from this region frequently seek TB treatment outside their home municipalities. This trend is attributed to limitations in the healthcare services provided by the municipalities, emphasising the importance of addressing these deficiencies to combat the spread of the disease effectively.A notable finding of this study is that it did not observe the achievement of the established 85% cure goal, and the dropout rate surpassed the threshold of 5%, indicating inadequate epidemiological surveillance in managing TB cases in this specific region. Crucially, the effective organisation of patient flow within the PHC in each municipality plays a vital role in fostering the dedication of health teams and facilitating coordinated care, ultimately leading to the achievement of TB control objectives. By establishing well-structured service networks, it becomes possible to analyse TB’s social, clinical, and epidemiological aspects, enabling the identification of risk groups and regions with a higher patient flow. This approach is precious in addressing disease outbreaks in vulnerable areas with neglected populations.


It is shown how the size, shape, and internal structure of the nanoparticles are related to the external laser ablation conditions, as well as to the laser melting processes of NPs in the colloidal solutions. This work provides important information about the generated nanoparticles, which can be used as building blocks for specially designed structures with predetermined optical properties.The application of a pulsed laser ablation technique for the generation of cerium-doped garnet nanoparticles in liquids is investigated. The morphological and optical properties of the obtained nanoparticles are demonstrated. Features introduced by the single crystals of Gd B and C atoms are coordinated by eight and six oxygen atoms, respectively [Garnets are a class of inorganic materials and have been known for many decades. The general chemical formula of garnet is Aectively ,5,6.In the past decades, there has been a fast expansion of nanotechnology and nanomaterials research, as well as their application in science and industry, which have raised the global demand for new advanced nanomaterials. Garnets in the nanoscale state usually feature higher luminescence intensity because of the light scattering and can offer controllable emission wavelength and improved efficiency depending on the size and type of the garnet material . Ce-dopeGarnet NPs have been mostly fabricated using chemical methods like sol-gel, solvothermal, co-precipitation, hydrothermal, combustion, photo-induced synthesis, etc. ,12. ChemTo improve NP purity, scalability, and reproducibility of the synthesis process, greater attention has been devoted to the research and development of physical synthesis methods. The application of the laser ablation technique to generate garnet NPs can avoid the disadvantages of chemical synthesis techniques and conforms to the principles of “green chemistry”. In addition, because molecular precursors are not required, the laser ablation technique is more cost effective compared to the chemical ones. Laser synthesis of nanomaterials can be classified into the following processes: ablation, fragmentation, and melting. Laser ablation is commonly used for nanomanufacturing, microstructuring, and cutting of materials and is often described as a high-temperature and high-pressure process. The laser generation of NPs has already become a separate scientific direction. Laser ablation can be performed in a vacuum, gases and liquids. The liquid is a relatively safe medium for the laser synthesis of NPs, compared to synthesis in an air environment where workplace contamination may endanger personnel. Pulsed laser ablation in liquids (PLAL) is a fast and efficient physical top-down technique where the use of liquids as a medium for the laser synthesis of nanomaterials makes it possible to control the efficiency of ablation by lowering the heat load on the target material and increasing the shock pressure on the surface. NPs synthesized using the PLAL technique are often preserved with the same chemical composition as the target bulk materials. Generally, a part of the ablated NPs is oxidized or reduced during PLAL, forming surface defects that can provide electronic stabilization. Such volume or surface defects can also be beneficial for optical applications .The PLAL technique requires laser setup, a target material in the form of bulk material or powder, and a liquid media. The size of the obtained NPs can be controlled using laser irradiation settings and parameters of the liquid media. Nowadays, most lasers have tunable parameters  that often affect each other. The wavelength of the laser beam for PLAL can vary between ultraviolet to near-infrared, depending on the transparency of the liquid medium. Pulse duration may be in the range from femtoseconds to milliseconds. Laser pulse repetition rate, by shortening and lengthening the time between pulses, affects the number of pulses per unit of time, the average delivered energy, and the pulse fluence. When the pulse energy is above the fragmentation threshold, smaller particles are produced due to explosion and evaporation, whereas when the pulse energy is below the fragmentation threshold, the NP’s size can increase due to melting processes . This ef−1 × cm−2) are used for the creation of explosive interactions with target bulk materials or particles for “top-down” particle production. On the other hand, for the PLML synthesis of nano- (1–100 nm) and submicrometer (100 nm–1 μm) particles, laser pulses with energy densities one or two orders of magnitude lower than for PLAL and PLFL are used (10–100 mJ × pulse−1 × cm−2). PLML is sometimes considered a modified PLAL technique with low-energy laser irradiation. In PLML, laser pulse interaction with particles leads to the aggregation of NPs and subsequent formation of larger particles, calling this method a “bottom-up” technique. As a result of an appropriately executed PMLM process, sphere-shaped particles are produced. But due to the uneven distribution of power within the beam, partially melted nanoparticles and agglomerates that consist of fused nanoparticles can also be generated  usually occurs, but also in other areas of the beam where the pulse power is several times lower. An additional laser melting of NPs can occur in those regions of the beam with low intensity. Such an effect is called pulsed laser melting in liquids (PLML) which has several differences from PLAL and PLFL. For PLAL and PLFL, ultrashort laser pulses with high energy densities  ,18. The The laser generation of NPs was conducted using the experimental laser setup, illustrated in –3 M, was obtained by dissolving a sample of Na3C6H5O7 × 3H2O in distilled deionized water . Sodium citrate water solution was chosen and used as a capping agent to prevent NP aggregation. Chloroform was chosen as a liquid medium for PLAL, taking into account the future potential applications in LEDs and scintillation materials in combination with 3D printing and other polymerization techniques. It is easier to incorporate garnet NPs, generated and stored in chloroform, into polymers that dissolve in chloroform like polymethyl methacrylate or polyvinyl butyral.Liquid parameters are crucial for the PLAL formation of NPs because of their influence on the size and mobility of cavitation bubbles, as well as on the local concentration of colloidal solution . PLAL in3Al2.4Ga2.6O12: Ce3+ (0.3 at. % Ce), Lu3Al5O12:Ce3+ (0.5 at. % Ce), and Y3Al1.25Ga3.75O12:Ce3+ (0.3 at. % Ce). Starting oxides Y2O3, Gd2O3, Lu2O3, Al2O3, and Ga2O3 had a purity no worse than 4N (99.99%). Powders for crystal growth were synthesized using a solid-state reaction under a temperature above 1600 °C in a neutral atmosphere for 10 h. Single crystals were fabricated using the Czochralski technique in an induction heating furnace “OXIDE” . GAGG:Ce and YAGG:Ce garnets that contained gallium were grown from iridium crucibles with a diameter of 60 mm and height of 60 mm under a weakly oxidizing atmosphere. LuAG:Ce was grown from a tungsten crucible with a diameter of 50 mm and height of 50 mm under an Ar + CO atmosphere. Growth parameters, such as pulling rate and rotation rate for each of the growth processes, were in the ranges of 1–1.5 mm/h and 10–15 rpm, respectively. As seed crystals, crystal fragments with crystallographic orientation relevant to (100) with square sections of 5 × 5 mm2 were used. To eliminate thermal stress after growth, single crystals were cooled down to room temperature during the 15 h wait and then annealed at temperatures above 1400 °C in an air atmosphere for 10 h. One side of every single crystal was polished for the homogeneous motion of the focused laser beam over the surface of the target material. Samples of each grown crystal are presented in For garnet NP generation, as target materials, single crystals of gadolinium aluminum gallium garnet doped with cerium (GAGG:Ce), lutetium aluminum garnet doped with cerium (LuAG:Ce), and yttrium aluminum gallium garnet doped with cerium (YAGG:Ce) were used. Garnet single crystals were grown from melts with the following stoichiometric compositions: Gd2 F20 TMP” from FEI . The UV-Vis absorption spectra were measured in quartz cuvettes using the “Shimadzu UV–1900i” double beam spectrophotometer .Laser-generated NPs were characterized using transmission electron microscopy (TEM) and UV-Vis absorption spectroscopy. High-resolution images and electron-diffraction patterns were measured with an analytical TEM “Tecnai GFor the laser generation of the NPs from every garnet single crystal, the laser setup was operated using the following parameters: λ ≈ 1064 nm; Δt ≈ 845 ns; repetition rate ≈ 560 Hz, pulse energy ≈ 212 µJ, with vertical polarization of the laser beam. For obtaining sufficiently concentrated colloidal solutions , Lu3Al5O12:Ce3+ (LuAG:Ce), and Y3Al1.25Ga3.75O12:Ce3+ (YAGG:Ce) single crystals that were grown using the Czochralski technique. Pulsed-laser-ablation generation was conducted in chloroform and a sodium citrate water solution. The synthesized garnet NPs have been characterized using transmission electron microscopy and UV-Vis absorption spectroscopy. All characterization methods indicated a less efficient laser generation in the sodium citrate water solution than in chloroform. Characterization of the morphology of the obtained garnet NPs showed signs of additional interaction of laser radiation with nanoparticles in a colloidal solution, which led to the formation of spherical particles, partially molten particles, and agglomerates of nanoparticles via the pulsed laser melting process. It has been observed that liquid media parameters have an impact on the NP’s shape and concentration in the colloidal solutions. A dependence of the average size of the synthesized NPs on the melting point of garnet single crystals has been observed. TEM images and diffraction patterns have shown that crystallinity is preserved in crystal debris, partially molten NPs, and agglomerates, but is absent in spherically-shaped NPs formed from completely molten crystal fragments. The UV-Vis absorption spectra of the NP colloidal solutions have shown an absence of Ce in all garnet NPs and the presence of Gd in GAGG:Ce NPs compared to the bulk materials.A comprehensive study of laser generation of garnet NPs has been conducted. Garnet NPs have been generated using the laser technique from Gd


Rps (Resistance to Phytophthora sojae) genes are used to manage Phytophthora sojae, a major oomycete pathogen that causes Phytophthora stem and root rot (PRR) worldwide. This study aims to identify temporal changes in P. sojae pathotype complexity, diversity, and Rps gene efficacy. Pathotype data was collected from 5121 isolates of P. sojae, derived from 29 surveys conducted between 1990 and 2019 across the United States, Argentina, Canada, and China. This systematic review shows a loss of efficacy of specific Rps genes utilized for disease management and a significant increase in the pathotype diversity of isolates over time. This study finds that the most widely deployed Rps genes used to manage PRR globally, Rps1a, Rps1c and Rps1k, are no longer effective for PRR management in the United States, Argentina, and Canada. This systematic review emphasizes the need to widely introduce new sources of resistance to P. sojae, such as Rps3a, Rps6, or Rps11, into commercial cultivars to effectively manage PRR going forward.Plant disease resistance genes are widely used in agriculture to reduce disease outbreaks and epidemics and ensure global food security. In soybean,  Rps genes are used to manage the major soybean pathogen Phytophthora sojae, which causes Phytophthora stem and root rot (PRR). Here, the authors show that widely used Rps genes are no longer effective for managing PRR in the United States, Canada and Argentina. Glycine max L.) is a major source of protein and oil that is produced on approximately 130 million hectares globally each year1. As the global population increases from 7.9 billion to an estimated 9.8 billion by 2050, we will need to produce more food on less land to ensure global food security2. The mission to increase the seed yield potential of soybeans has been at the forefront of soybean breeders for decades, and consequently, present soybean varieties produce nearly twice the seed yield they did 60 years ago3. However, efforts to achieve yield gains through genetics are often at the expense of decreased levels of disease resistance4.Soybean (Phytophthora sojae (Kaufmann & Gerdemann), is responsible for 1–2 billion U.S. dollars in soybean yield loss worldwide5. Outbreaks of PRR are more prevalent after heavy rains in fields where soil moisture remains saturated and facilitates the production of motile zoospores of P. sojae, which move chemotactically toward soybean root exudates and infect soybean roots. During infection, the sexual reproductive and resting structure, the oospore, is produced within infected soybean root and stem tissue6. P. sojae is homothallic, or self-fertile, and can produce viable oospores without the need for a second genotype. Oospores of P. sojae can lie dormant within plant debris in the soil for years until environmental conditions are conducive to infection  and susceptible host plants are present7.Phytophthora stem and root rot (PRR) of soybean, caused by the soilborne oomycete 8. The increasing frequency of heavy rains could provide more favorable environmental conditions for PRR disease throughout the global soybean-producing regions, emphasizing that effective and economically viable management of this destructive soybean disease will be increasingly important9.Current climate models suggest that periods of intense rain may become more frequent due to anthropogenically driven climate changeP. sojae resistance genes  and quantitative resistance are commercially available to mitigate losses to PRR specifically. The first Rps gene available for PRR management was Rps1a, released in 1964, followed by Rps1c in 1980, and lastly, Rps1k and Rps3a, released in 198510. The genes Rps1c, Rps1k, and Rps3a were made commercially available to manage PRR after Rps1a-mediated resistance in Ohio became no longer effective in 197210. Given the adaptation of the P. sojae population to evade Rps1a-mediated resistance over 8 years, Schmitthenner hypothesized that Rps genes would only be effective for management for 6 to 15 years before needing to be replaced by more efficacious Rps genes11. This observation raises current concerns for the efficacy and longevity of Rps1c, Rps1k, and Rps3a for PRR management on a global scale, as they have been deployed for 35–40 years10. Currently, more than 40 Rps genes have been identified in soybean germplasm; however, only a few are widely effective and released for PRR management12. In North and South America, Rps1c and Rps1k are the most widely available Rps genes to farmers for PRR management, while the availability of Rps1a, Rps3a, Rps6, and Rps8 depends on country, locale, and seed company13.Currently, soybean varieties with single-dominant Rps proteins initiate strong qualitative defense responses against P. sojae isolates expressing specific Avr proteins (effectors). This gene-for-gene relationship has been well characterized and is the primary means of disease management for PRR in agricultural production worldwide16. Soybean Rps genes are thought to encode for NBS-LRR (Nucleotide Binding Site–Leucine Rich Repeat) receptor proteins which recognize Avr proteins or their activity from P. sojae. When P. sojae produces Avr proteins detected by the complementary soybean Rps protein(s), genetic signals are produced that lead to a hyper-sensitive response that confers complete resistance to P. sojae17. Alternatively, P. sojae isolates that do not express effectors recognized by the cognate soybean Rps protein evade detection, continue infection, and cause PRR. A standard set of soybean germplasm, which differs in the Rps gene present in the genome, is used to characterize the gene-for-gene interaction phenotypically and identify effective Rps genes in the sampled P. sojae population18. Recently, new molecular tools have been developed to rapidly identify Avr genes present in P. sojae isolates and thus assess the pathotype profile without going through the phenotypic procedures20. Through surveying populations of P. sojae and describing their pathogenicity phenotype, effective resistance genes for the management of the sampled population can be identified and recommended for use by commercial producers.Soybean P. sojae populations has occurred in North America since the first pathotype of P. sojae was described in 196541. While P. sojae was identified and known to be prevalent in South America since the 1970s, surveys have only been conducted since the late 1980s46. Surveys in Asia, primarily China, where P. sojae is an invasive pest, have been performed since 1991, when the pathogen was first identified in Heilongjiang Province, China54. Surveys in other soybean-growing countries such as Japan, Iran, and Australia have been infrequently conducted58. These surveys typically were performed on a state or province scale and occasionally aggregated to offer insights into Rps gene efficacy on a regional scale at a single time point49. In addition to identifying effective Rps genes, these studies have also been used to describe and track the pathotype complexity, the number of Rps genes which isolates can cause disease against, as well as the diversity of pathotypes to better understand the durability of Rps-mediated resistance. As the pathotype complexity of the P. sojae population increases, there will be fewer individual Rps genes that can effectively manage the known population and stacking, or having multiple Rps genes present within the genotype, will be needed for effective disease management. Likewise, observations of increasingly diverse pathotypes mean it is highly unlikely these populations would be effectively managed through the use of a single Rps gene. Pathotype complexity and diversity metrics can be used to assess the potential durability of employed Rps genes in a given population. Pathotype evaluation surveys at multiple timeframes in the same regions have increased concerns about increasing pathotype complexity and diversity, and the potential loss of effectiveness of the Rps1c and Rps1k genes for PRR management in North and South America have emerged45. However, no comprehensive worldwide analysis has been performed to evaluate the durability of Rps genes over time and across continents, which would allow for more informed planning and decision-making in global commercial trait-introduction programs to adequately manage PRR in the field.Regional or state sampling of P. sojae pathotypes have evolved, as well as the durability of Rps genes for PRR management, will help guide soybean breeding and disease management recommendations on a global scale. Here, we use a novel approach of collating data from the past thirty years of P. sojae pathotype surveys from four of the largest soybean-producing countries  across three continents to show the first global-scale Rps gene fluctuation. Specifically, we aimed to: (1) identify countrywide-temporal changes in pathotype complexity, (2) evaluate Rps gene efficacy on a country-temporal scale, and (3) determine how the pathotype diversity of sampled P. sojae populations have changed over time by country. We determined that: (1) the pathotype complexity of P. sojae populations in Argentina, China, and the United States has significantly increased over time, with sampled populations now able to overcome 1–3 more of the tested Rps genes than in previously sampled time frames, (2) Rps1c and Rps1k are no longer effective in the United States, Argentina, and Canada; however there is little change in Rps1c and Rps1k efficacy observed in China between the time frames studied, and (3) the diversity of pathotypes has significantly changed over time in each country, leading to inadequate PRR disease management using currently employed Rps genes. Together, these results broadly build upon foundational research on the management of PRR. Moreover, they significantly improve our current understanding of Rps gene efficacy over time and provide a rationale for deploying new Rps genes and quantitative resistance to ensure adequate management of PRR globally.Identifying how regional Phytophthora sojae pathotype composition within populations is thought to evolve and change over time due in part to the selective pressure of always planting soybean varieties containing the same Rps gene each rotation10. A P. sojae population adapts due to this constant selective pressure from the host through mechanisms such as outcrossing with other P. sojae genotypes59, as well as mutations61 or gene-silencing62 of the detected P. sojae Avr-gene product. If the soybean Rps gene is unable to detect infection by P. sojae, no defense response is activated; the plant is therefore susceptible to infection, and disease ensues. In total, 41 P. sojae pathotype studies were identified through our search of the literature. Of these, 29 were used for this systematic review  that have been used to describe isolate pathotypes in P. sojae pathotype surveys for decades22. In soybean production, Rps1c and Rps1k are currently the most commonly deployed genes available in cultivars for the management of PRR, while Rps1a, Rps3a, and Rps6 are available to a lesser extent13. We did not include Rps2, Rps3b, Rps3c, Rps4 and Rps5 in this analysis, as not all studies used these resistance genes to characterize their P. sojae pathotype composition of field populations, and none of these Rps genes are used for management of PRR in commercial soybean production in these nations.Rps genes an individual P. sojae isolate within the population can cause disease against. Thus, these genes are ineffective for PRR management purposes. The published pathotype data from Argentina, Canada, China, and the United States were grouped into two or three distinct timeframes  for all temporal analyses, depending on the availability of data from each country , Canada , and the United States  between the 2000–2012/2013 and 2013–2019 timeframes  increase in pathotype complexity was observed between the two timeframes, with a mean increase of 0.317 Rps genes , Rps1c (66.6%), and Rps1k (78.1%). More than 75% of the isolates tested were pathogenic on Rps1a and Rps1k , Rps1c (74.5%), and Rps1k (85.5%). Likewise, increases in pathogenicity between the 2000–2013 and 2013–2019 timeframes on soybean genotypes containing Rps1a (from 60.9 to 89.1%) and Rps1b (from 44.1 to 83.5%) were also observed. Interestingly, the efficacy of Rps3a and Rps6 genes on the United States P. sojae national population appears to have remained consistent throughout the past thirty years  and 2014–2019 (52.5%), however, population pathogenicity on Rps6 decreased from 55.7 to 11.9%. Rps1d is not widely deployed in Canadian soybean production13; it is curious why the sampled population is adapting to this resistance gene on a national scale. Of the Rps genes tested with the population from China, only the efficacy of Rps6 changed between 1990–1999 (26.8%) and 2000–2013 (58.9%). The efficacy of the other tested Rps genes remained consistent throughout the timeframes used for analyses  and Principal Coordinates Analysis (PCoA), in tandem with testing beta-dispersion and Permutational Multivariate Analysis of Variance (PERMANOVA) to determine significant shifts in these populations over time. The Gleason diversity index exhibited an increase in pathotype diversity between the first and last timeframes for the P. sojae populations in the United States, Canada, and China , and began to coalesce during the 2013–2019 time frame as compared to the 2000–2013 time frame . Similar findings were obtained in Argentina between the 1989–1999 and 2000–2012 time frames , as well as the 2000–2012 and 2013–2019 time frames . Due to the availability of data, the change in pathotype beta-diversity could only be compared between 2000–2013 and 2014–2019 in Canada , and between the 1990–1999 and 2000–2013 time frame in China, of this study . However, the results from Canada and China corroborate what was found in the United States and Argentina during their respective time frame. Reductions in beta-dispersion over time indicate that more isolates are becoming similarly virulent on a number of the Rps genes tested. Beta-dispersion and PERMANOVA tests corroborated alpha-diversity test results; there was a significant difference in each country’s pathotype composition between sampling timeframes, with pathotypes becoming less dispersed over time . Following the hypothesis made by Grijalba and Gally15, pathotype complexity and diversity continued to increase through the 2000s while effective resistance genes in the region decreased. Nevertheless, the loss of Rps1c and Rps1k for management of PRR in North and South American commercial soybean production presents an immediate concern for global food security.While the loss of the P. sojae are diverse within populations, and single Rps gene-mediated management will not be an effective long-term management strategy going forward unless new and widely effective Rps genes are deployed into commercial soybean varieties on a regular basis. In Canada, it was found that 85% of soybean growers were using an ineffective Rps gene within their fields, showing the need for novel and effective Rps genes13. Reports from the United States estimate that effective Rps genes such as Rps3a and Rps6 were present in less than 3% of soybean varieties from 2010 to 202037. The efficacy of Rps3a and Rps6 remaining relatively stable during the past thirty years in the United States is likely due in part to their very limited availability to producers and therefore minimal selective pressure on the P. sojae populations studied here  have been identified in soybean for resistance to P. sojae, along with their associated markers for marker-assisted breeding, which are available in Lin et al.12.Quantitative resistance  to Rps genes used for PRR management in major soybean-producing countries of North and South America, Rps1c and Rps1k, are no longer effective, and alternative forms of resistance will be needed to manage PRR going forward. Concentrating breeding efforts on introgressing quantitative resistance into commercial soybean varieties, in conjunction with stacking and deploying novel Rps genes, may reduce the impact of future epidemics of PRR on global food security in these countries.A conclusion of this systematic review is the predominant 87. This systematic review has not been registered in the International Prospective Register of Systemic Reviews (PROSPERO), and the protocol has not been implemented or published before. The PRISMA checklist for this manuscript can be found in the supplement , Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps3a, Rps6, Rps7) to ensure homogeneity of phenotypic observations for each isolate. The Rps7 data from Canada was excluded as the most recent survey used a molecular tool to detect P. sojae Avr genes, and Rps7 testing was not performed13. All other pathotype surveys used the soybean hypocotyl inoculation technique for pathotype characterization, as described in Dorrance et al.68. P. sojae isolates data were then grouped by country and timeframes for analysis by the year each sample was reportedly isolated from soil or from symptomatic plants. Data from studies that were outside the discrete timeframes examined were not used in the analysis, and no isolate data was used in more than one timeframe (Table n = 1115), 2000–2013 (n = 956), 2013–2019 (n = 1067) , 2000–2012 (n = 65), 2013–2019 (n = 210) , 2014–2019 (n = 394) , 2000–2013 (n = 790)  was determined for each individual P. sojae isolate by country before calculating the mean pathotype complexity and standard deviation of a sampled P. sojae population at each timeframe and country. A Welch’s two-sample, two-sided t-test was used to determine the significance between pathotype complexity at each timeframe by country.Using the ‘hagis’ R package, pathotype complexity ; percent pathogenic = ((‘number of isolates pathogenic’)/)*100. Percent isolate pathogenic results were plotted using ‘ggplot2’90 (Version 3.3.5) with a dashed red bar at the 40% management efficacy threshold to show effective and ineffective Rps genes for each of the sampled population timeframes. Forty percent was used as a conservative estimate for management efficacy, as it is before the majority of the population would be able to cause disease against a given Rps gene.Pathotype data for each country was grouped by timeframe, and the number and percentage of isolates pathogenic on each 88. The Gleason diversity index is calculated as: Gleason = ((N unique pathotypes) − 1)/log(number isolates)91. The ‘hagis’ R package automatically removes non-pathogenic phenotypic data from the dataset before calculating diversity indices so that only differences in pathogenicity are described.The Gleason diversity index, and number of unique pathotypes, were calculated for country timeframes using the ‘hagis’ R packageP. sojae isolates which were non-pathogenic on all of the 8 Rps genes, and the susceptible (rps) control phenotype was removed. Jaccard distances were calculated for each country individually using the ‘vegan’ R package92 (version 2.5–7). Principal Coordinates Analysis was performed using the Jaccard distance matrix for each country and visualized with 95% data ellipses from the ‘ellipses’ R package93 (Version 0.4.2) using ‘ggplot2’90 (Version 3.3.5). Beta-dispersion tests were conducted between timeframes using the function “betadisper” from the ‘vegan’ package92. An Analysis of Variance (ANOVA) was used to identify significant differences in Beta-dispersion between timeframes, and the ad-hoc Tukey’s HSD test was used to determine significant pairwise differences between groups. A Permutational Multivariate Analysis of Variance (PERMANOVA) was performed using “adonis”, with 999 permutations, from the ‘vegan’ R package to test for significant differences in the pathotype composition of the sampled populations within each country between timeframes. PERMANOVA significance was further investigated using a pairwise PERMANOVA from the ‘RVAideMemoire’ R package94 (Version 0.9-81-2) with the function “pairwise.perm.manova” with 999 permutations.To make statistical comparisons between pathotype diversity for each timeframe, the data from each country was further reduced to exclude Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource data file


Existing food trends and modern consumers’ nutritional preferences have led to a rising demand for plant-based sources of protein such as hemp seed meal and the possibility of consumption hemp-rich products, most often in wheat-based staple foods, such as pasta. Pasta, as a conventional food product, is widely consumed worldwide due to its nutritional value, long shelf life, easy preparation, versatility of uses and also relatively low cost, which has made this product popular over time. Five formulations of rigatoni-shaped pasta obtained by partial replacement of wheat grain flour with 5%, 10%, 15% and 20% hemp seed meal (HSM) were studied regarding the technological, physicochemical, textural, antioxidant and sensory properties of the pasta samples. The substitution of wheat flour with hemp seed meal (HSM) led to a slight increase in the cooking loss (CL) and optimal cooking time (OCT) compared to the control sample, while the water absorption (WA) and swelling index (SI) decreased during evaluation. The experimental results also showed a decrease in luminosity and fracturability, with an increased firmness of pasta dough. Moreover, the developed pasta showed a significant improvement in antioxidant capacity in terms of total phenolic content (TPC) and antioxidant activity (DPPH). The pasta samples with 15% and 20% HSM substitutes experienced a browning process due to the Maillard reactions during drying, as well as a color loss during cooking; however, the color changes did not affect the acceptability of the product. The partial replacement of wheat flour with hemp seed protein highlighted the possibility of developing a new innovative type of pasta that claims a functional benefit and presents an improved nutritional value, mainly due to the partial protein intake, as well as certain benefits for a human diet. Globally, pasta consumption and sales reached USD 22.8 Billion in 2022, and this value is expected to grow fluently as consumers are more conscious of the environment and its major climate changes, but also the trendy adoption of a vegan or vegetarian health-promoting diet significantly decreased the demand for meat-based food products . Pasta rFor the functional foods segment—which includes such products as nutrition bars, meal replacers and supplements, and innovative food products in general—protein content and its quality is one of the most important requirements .As the nutritional needs of the consumers are mostly focused on the quality of those amino acids that a plant-based protein can provide, hemp seed protein can be considered one of the most biologically suitable protein substitutes for a meat-type protein . ChemicaTherefore, hemp-rich products, especially pasta enriched with hemp seed meal (HSM) contribute to a healthy, complete and nutritionally balanced diet, helping to maintain your body and keep your mind active.The importance of this research is reflected in the consumer’s demand oriented towards functional products and protein-enriched products. Moreover, the work supports food processors with useful information related to the technological behavior of the dough and hemp pasta samples while cooking, as the final product quality depends on its behavior during processing. Although the nutritional needs of the consumers can be satisfied by formulating a new type of functional pasta, the feasibility of the product itself might suffer some significant difficulties, especially in terms of technological properties and several noticeable consequences of heat processing .The present research aims to study the effect of the partial replacement of wheat grain flour with hemp seed meal (HSM) on a new assortment of pasta, which has a functional benefit, regarding the technological, rheological, textural and sensory properties of pasta samples.The main raw materials used for rigatoni pasta were wheat flour of 650 type  and hemp seed protein powder  which was produced by grinding down to a fine powder the hemp seed “meal” also called hemp “cake”, a by-product resulted from hemp oil extraction . AccordiWheat flour was substituted with different levels of hemp seed meal (HSM) as following: 0%—control sample, 5%—HSM_5, 10%—HSM_10, 15%—HSM_15, 20%—HSM_20. The dough of the pasta samples was formulated by mixing all ingredients, using a Kitchen Aid  mixer. The hydration levels used were 58.7% for the control sample, 59.9% for HSM_5, with 5% wheat substitution, 60.4% for HSM_10 (10%), 61% for HSM_15 (15%) and 61,4% for HSM_20 with 20% of wheat substituted with hemp seed meal (HSM), respectively. Each sample was also made using 2 g of salt The process itself included several steps: first, the mixing of all solid ingredients, such as wheat flour, hemp seed meal and salt, followed by adding water and mixing again for 3–5 min using a dough hook until all ingredients were evenly combined. The resulted firm dough ball was extruded using a KitchenAid pasta press with a rigatoni mold. Rigatoni-shaped pasta was dried in a functional dryer with a heat ventilator unit to ensure uniform temperature and ventilation in all parts of the dryer and a moisture control unit for the constant humidity at 40 °C for 16 h. After a complete drying circle, pasta was cooled for 2 h at room temperature and subsequently stored in plastic bags with zip locks until further experiments were performed.In order to obtain the rheological extension data of pasta dough, an Alveograph device  was used following the standard methods procedures AACC 54-30A, ICC 121 or ISO 5530/4. In essence, a mixture of wheat flour, hemp seed meal and salty water mixed into a dough ball is supposed to resist a biaxial extension, which is generated by a pneumatic circuit, forming a rising bubble of dough due to the gluten network, which is continuously filling with air until it breaks at a critical point/value. The following parameters were measured: dough extensibility (L), baking strength (W), maximum pressure (P) and the configuration ratio of the Alveograph curve (P/L ratio) of the pasta dough .i) and maximum gelatinization temperature (Tmax) were analyzed.The dynamic dough rheological properties were evaluated using a Thermo-HAAKE, MARS 40 rheometer . The frequency sweep test was performed on laminated dough samples, rested in advance for 30 min at room temperature for internal strain elimination. Storage modulus (G’) (elastic property), loss modulus (G”) (viscous property) and loss tangent (tan δ) were evaluated. Each pasta sample prepared to the optimum dough hydration level was analyzed using two parallel plate geometry (d = 40 mm) and a gap of 2 mm. During the sweep test, frequency varied from 0.1 to 20 Hz, at a constant strain of 15 Pa. For the temperature sweep test, the dough samples were heated from 25 °C to 100 °C at a rate of 4 °C per min. For this purpose, the initial gelatinization temperature (TThe morphology of the dry pasta surfaces was determined using a scanning electron microscope (SEM) Hitachi SU-70  equipped with a detector of secondary electrons (SE) of the Everhart-Thornley type, which requires a high vacuum environment. The samples were metallized with a layer of 5 nm of Ag. The acceleration voltage was 5.00 kV, and the images were obtained at a 1000× magnification.The technological parameters of the rigatoni pasta samples that give a better appreciation of pasta cooking quality were determined by the AACC International Approved Method 66-50.01 . All pasThe optimal cooking time (OCT), expressed in minutes, was determined according to the AACC International Approved Method 66-50.01 . For OCT1 is the weight of the cooked pasta and w2 is the weight of the pasta after drying [An amount of 10 g of pasta sample were boiled in 200 mL of distilled water for a certain period of time equivalent to the optimal cooking time (OCT) of each pasta sample, followed by draining the excess water using a fine sieve for 3 min and weighting the resulted cooked product. The water absorption index was determined with Equation (1):r drying .Cooking loss was determined using the water collected from the previous determination of water absorption index (WAI) by drying the whole amount of collected water in a heat-resistant glass vessel in an oven at 105 °C until total liquid evaporation and weighting the solid residue. Cooking loss (CL) represents, therefore, the amount of solids lost into the cooking water, expressed in grams/100 g of raw pasta .1 is the weight of the cooked pasta and m2 is the weight of the pasta after drying [Swelling Index was obtained by drying cooked pasta, previously boiled according to its OCT, until a constant weight at 105 °C (approximately 16 h), then cooled at room temperature and weighed again. The final results were calculated with Equation (2):r drying .CIEL*a*b* color space was used, where in terms of colorimetric coordinates, L* represents the lightness while a* and b* represent the green to red and blue to yellow color ranges, respectively [Dry and cooked pasta color parameters were measured using a Minolta Chromameter . In order to obtain precise and consistent color evaluation ectively .Pasta textural parameters were performed using a texture analyzer TVT-6700  equipped with a 10 kg load cell, as in the method described by Iuga, 2020 . An amouDry pasta was submitted to a fracturability test that measured the maximum force F (g) required to break a dry pasta sample into multiple pieces using a Perten TVT-6700 device . For this purpose, an aluminum break rig set was use, adjusted to 13 mm width, with a trigger force of 50 g at a speed set to 3 mm/s . For thev/v) in a sonication bath for 40 min at 37 °C and 45 kHz, followed by a double filtration. The determination of total phenolic content (TPC), expressed in μg GAE/g, was made using the Folin–Ciocalteu method as follows: 0.2 mL of extract (diluted 1:5 with distilled water) was mixed with 2 mL of Folin–Ciocalteu reagent (1N) (diluted 1:10 with distilled water) and 1.8 mL of sodium carbonate 20% (w/v) into a tube. The solution was left in the dark at room temperature for 30 min. The TPC determination was performed at 750 nm wavelength on a UV–VIS–NIR Shimadzu 3600  spectrophotometer. Each sample was evaluated in triplicate, and TPC values were calculated through extrapolation using a calibration curve (R2 = 0.99) made with different concentrations of gallic acid (GAE).Extraction and determination of total phenolic content (TPC) were performed according to the methods described in ,27, on ps is the absorbance of the sample and Ab is the absorbance of the blank sample, both read at the same wavelength (λ = 517 nm).Pasta antioxidant activity was assessed by reading the absorbance of the solutions at 517 nm wavelength using a UV-VIS-NIR spectrophotometer, as in the method described by Chetrariu , as follThe sensory profile of the pasta samples was evaluated using 30 semi-trained panelists. The sensory evaluation was performed at the Sensory Laboratory Lab of the Faculty of Food Engineering, Stefan cel Mare University, Suceava, Romania. Each panelist evaluated the sensory characteristics of the pasta samples, which were placed on white plates codified with black three-digit numbers, which did not contain the letter A or the number 1. Before tasting, each panelist cleaned their palate with water. For sensory analysis, the hedonic test was used with a nine-point hedonic scale (1—“dislike extremely” to 9—“like extremely”). The sensory pasta characteristics evaluated were texture, taste, smell, color, aroma, aspect and general acceptability.p < 0.05) [All experimental data obtained were processed using the Statistical Package for Social Science . The experimental data were expressed as the mean value ± standard deviation. The statistical differences between means were analyzed by applying ANOVA analysis of variance, with Tukey’s test at a 5% significance level ( < 0.05) . The relThe wheat flour that was used for pasta formulations presented the following data: 0.65% ash content, 1.17% fat, 30.2% wet gluten, 11.9% protein, 14.2% moisture, 4 mm gluten deformation index and 386 s the falling number value. According to the wheat flour data value characteristics, this was of a strong quality for bread making and low α-amylase activity . The hemp < 0.05) between Alveograph values maximum pressure, extensibility and P/L ratio. All the dough samples presented high P/L values (˃1), indicating a strong gluten suitable for pasta production [The empirical dough rheological characteristics during the extension of pasta samples are shown in oduction . From aloduction . Due to The fundamental rheological characteristics of dough samples formulated by partial replacement of wheat flour with different levels of HSM can be seen in SEM Analysis allows a better understanding of the textural profile of the pasta samples and helps to appreciate the effect of the partial replacement of wheat grain flour with different levels of hemp seed meal on the dough strength, which influences the quality of the final product . Figure p < 0.05) of the OCT value compared with the control sample but still presented a lower value compared to the manufacturing rigatoni OCT parameters, which were between 11 and 14 min [p < 0.05) compared to the control sample.The pasta technological characteristics, in terms of optimal cooking time (OCT), water absorption (WA), cooking loss (CL) and swelling index (SI), are shown in d 14 min . This isd 14 min ,13. Fromd 14 min . A variad 14 min , as welld 14 min . The parp < 0.05) in brightness (L*) and an increase in redness , which may be associated with the development of the Maillard reaction [Pasta color is one of the most important sensory characteristics that considerably influence consumers’ purchasing decisions. At the same time, the color of the food product is unconsciously associated with its aroma and probable taste. The changes in pasta color parameters after the partial substitution of wheat flour with 5%, 10%, 15% and 20% HSM in dry and cooked pasta, which can be seen in p < 0.05) were noticed for cohesiveness and stickiness values when high levels of HSM were incorporated into the wheat flour. Compared to the control sample, the firmest pasta sample was the one with 20% HSM incorporated in the dough recipe. In addition, this sample presented the lowest adhesiveness value. Similar data have also been reported by Chetariu and Dabija when spent grains were incorporated into a dough recipe [h recipe .The partial replacement of wheat flour with HSM showed a decrease in fracturability compared to the control sample, as can be seen from p < 0.05) compared to the control sample. This fact was explainable because hemp seed is rich in natural antioxidants and phenolic compounds [ompounds . Similarompounds  or gluteompounds  in whichThe obtained data for sensory characteristics are shown in p < 0.05) the protein, fat and ash of the pasta samples, whereas the carbohydrate content decreased. This fact is explainable because HSM presents more fat, protein and ash compared to the wheat flour sample.The nutritional value of pasta samples formulated by partial replacement of wheat flour with different levels of HSM is shown in The associations between characteristics and pasta samples can be seen in The first principal component explains 57.65% of the total variance, whereas the second explains 20.97%. PC1 was closely associated with pasta technological characteristics, color data of dry and cooked pasta, loss tangent, sensory characteristics except for aspect, textural characteristics except for cohesiveness and nutritional pasta characteristics, while PC2 was associated with dough empirical rheological properties during extension and dynamic module ones, cooking loss, moisture, cohesiveness, sensory pasta and aspect. Pasta samples that contain high levels of HSM substitutes of wheat flour are closer related to each other by being located on the right side of the PCA graph, while the control sample and HSM_5 are placed on the left part of the graph alongside the PC2 component on the PCA. The placement of control sample and HSM_5 on the PCA plot indicates that these pasta samples are very distinctive compared to the samples with higher levels of HSM in the pasta recipe.p < 0.05) the dough’s empirical rheological properties during extension. At low levels of HSM enrichment, dough has more strength due to an increased value of tenacity and baking strength. This may be because HSM presents a high protein amount, which may prevent the complete hydration of the rest of the dough compounds, mainly starch. In addition, besides proteins, HSM contains other components that may change water binding, affecting the dough rheological properties. Similar data have been reported by Korus et al. for bread and cookie dough where hemp seed proteins were used [p < 0.05) for the samples with 5% and 10% HSM incorporated into the dough recipe. These data are in agreement with those reported by others who have also concluded that dough rheological characteristics are not significantly different between samples with these levels of hemp meal enrichment [Compared to the control sample, hemp protein flour significantly changed  and higher redness . Such parameters were obtained due to the Maillard reactions, as the process of drying requires relatively high temperatures and the process itself takes several hours. For pasta with 20% HSM, the drying and cooking heat treatments led to significant changes of the pasta color. During sensory evaluation, panelists noticed an unpleasant color, which influenced the general acceptability of the product.The same as in the case of pasta enriched with hemp raw materials, as described by Teterycz et al., 2021 , the parSensory profile evaluation confirmed the interest of consumers regarding the innovative pasta formulations, as panelists noticed the appealing green color and pleasant taste of HSM_15 pasta sample with wheat flour substituted with hemp seed meal in proportions of 15%, which presented the optimal sensory parameters. On the other hand, lower levels of HSM negatively influenced the panelists’ perception of the product, as they noticed the color loss, an unpleasant stickiness of the samples while chewing and pasta samples were also tasteless compared to the higher levels of HSM enrichment.p < 0.05) its total protein, fat content, phenolic content and antioxidant activity. The nutritional improvements of cereal products in which hemp seed meal have been incorporated have also been reported by others [From a nutritional point of view, HSM-enriched pasta significantly increased  were obtained between dough extensibility and index of swelling (r = 0.99) due to the fact that both are expressions of dough rheological properties during extension [p < 0.05) with textural pasta characteristics firmness (r = −0.944), stickiness (r = 0.926) and pasta fracturability (r = 0.890). This association may be due to the gluten dilution in the dough system from using the HSM, a non-gluten ingredient that partial substitutes wheat flour in the pasta recipe. Total phenolic content and antioxidant activity are closely significantly (p < 0.05) associated with each other (r = 0.955), being positively (p < 0.05) correlated with fat  ash  and negatively (p < 0.05) correlated with dry pasta luminosity , pasta’s luminosity  and carbohydrates content . The HSM replacement in wheat flour increased the pasta nutritional characteristics due to its much higher protein, ash, fat content, antioxidant activity and total phenolic content compared to wheat flour. Therefore, these characteristics are significantly correlated. In addition, HSM presents natural pigments that obviously change the pasta color properties [The PCA plot shows a clear dissimilarity between the pasta samples. The control sample positioned at the bottom left of the graph is closely related to swelling index, water absorption, fracturability, adhesiveness, pasta luminosity and sensory characteristics such as smell, taste and texture. This sample is closely related to the pasta sample with the lowest level of HSM replacement in wheat flour, whereas the samples with high levels of HSM incorporated into the pasta recipe are more closely associated with being more related to the nutritional characteristics of pasta samples. Significant correlations  and antioxidant activity of enriched protein pasta confirm the functional potential of the product, as the consumption of hemp seed-supplemented products is associated with reducing the risks of multiple stress oxidative diseases. From a sensory point of view, HSM-enriched pasta was well appreciated, as panelists noticed an appealing dark green color and pleasant taste of the product (HSM_15).The experimental data obtained in this study will definitely be useful for other researchers, as the hemp meal use in different food products is currently not studied enough but represents a high interest topic worldwide. The experimental results will also be used in our further investigations.


The prevalence of factor VII deficiency (F7D) is 1 in 500,000. Due to its rarity, the management of bleeding disorders in pregnancy is not well established. We examine a case of an 18-year-old  woman at approximately 19 weeks gestation with a known history of F7D who presents after a motor vehicle accident. Fetal demise was confirmed necessitating a medical induction. She also had multiple fractures requiring surgical intervention. A multidisciplinary team consisting of orthopedic surgery, obstetrics and gynecology, and hematology/oncology was consulted for optimal timing of factor VII replacement prior to procedures. The patient underwent successful left tibial intramedullary nailing with minimal bleeding. She received factor VII and tolerated an uncomplicated vaginal delivery. Her postpartum and postoperative courses were uncomplicated, requiring one unit of packed red blood cells. The patient was discharged on postpartum day three. Management of this second-trimester abortion with a history of F7D was possible with effective communication and the organization of a multidisciplinary team to account for the risk of thrombosis versus hemorrhage and the availability of factor VII replacement therapy. Factor VII deficiency (F7D) is an autosomal recessive rare inherited coagulation disorder (RICD) affecting the extrinsic pathway of the coagulation cascade. The prevalence of F7D is 1 in 500,000 and 1 in 2 million for homozygous forms. Due to the rarity of RICDs, the type and severity of bleeding symptoms, underlying molecular defects, and management of bleeding disorders are not well established . This caThe patient is an 18-year-old  woman at 19 weeks and 4 days gestation who presented to the emergency department after a motor vehicle accident. As the restrained driver, traveling at approximately 13.4 m/s, another vehicle going at approximately 22.4 m/s collided with the patient’s passenger side of the vehicle. Airbags were deployed, and the patient was extricated from the vehicle. The patient’s Glasgow Coma Scale was 15, and on physical exam, she was normotensive, without signs of hemorrhage, and oxygen saturation was in the high 90s on room air. Primary and secondary surveys were completed, and no immediate intervention was required. Bedside Focused Assessment with Sonography for Trauma was negative, except for weak fetal cardiac activity. A formal obstetric ultrasound was ordered confirming placental abruption and fetal demise. Imaging was significant for comminuted displaced fracture of the left mid tibia and fibula, left clavicle fracture, and a small left pneumothorax.The patient was diagnosed with F7D at the age of two after an episode of epistaxis. She reported a history of heavy menstrual bleeding but otherwise denied other sources of spontaneous bleeding. The patient had previously undergone an appendectomy and tonsillectomy without hemostatic complications. According to her hematologist, she was heterozygous F7D, and her last known levels were 40%. Other family, maternal, and obstetric histories were unremarkable.The management of this patient’s care involved the collaboration of various specialty teams including emergency medicine, trauma surgery, obstetrics and gynecology, orthopedic surgery, critical care, hematology/oncology, pharmacy, physical therapy, occupational therapy, and rehabilitation services. A multidisciplinary team meeting was held to assess the timing of orthopedic surgery versus medical induction of labor and the risk of thrombosis versus hemorrhage. The patient underwent left tibial intramedullary (IM) nailing with orthopedic surgery on hospital day two (HD#2) and tolerated the procedure well with minimal bleeding. The patient did not require operative management of her left clavicle fracture. Hematology/oncology was consulted in preparation for medical induction of labor who recommended the administration of one dose of coagulation factor VIIa at 40-50 mcg/kg (approximately 2 mg) every 12 hours to 24 hours with frequent monitoring prior to the procedure. They also recommended against the use of low molecular weight heparin (LMWH) prior to delivery as it counteracts the hemostasis efforts. The patient was at increased risk of clotting, stroke, deep vein thrombosis, and pulmonary embolus (PE) with factor VII replacement but also paradoxically at high risk for postpartum hemorrhage from F7D. She received misoprostol 800 mcg followed by 400 mcg three hours later which successfully led to an uncomplicated medical abortion on HD#3 in the ICU. There was no evidence of fetal movement, breathing, or pulse, and fetal demise was confirmed. The estimated blood loss was 200 ml. Placental pathology showed a 4-cm retroplacental hematoma and separate fragments of clotted blood, consistent with placental abruption. Her postpartum course was uncomplicated; thus, the decision was made to start the patient on LMWH after she received coagulation factor VIIa. The pneumothorax resolved spontaneously. Her hemoglobin was 6.6 gm/dL on HD#4 after the medical abortion which was a drop from 11.0 gm/dL on the initial presentation. She received one unit of packed red blood cells (pRBC), and her hemoglobin improved to 9.4 gm/dL on HD#5. Four doses of coagulation factor VIIa 2 mg were given to the patient in total. The patient was monitored and remained stable for subsequent discharge on HD#5, postpartum day three (PPD#3). Table Given the emergent nature of this patient’s presentation with a history of F7D, one requires an understanding of the disorder with a systematic approach through a thorough history and physical examination in collaboration with various specialty services to prevent hematologic emergencies. This patient is a heterozygous carrier of F7D resulting in a quantitative deficiency where plasma levels of the factor are reduced. Generally, heterozygous gene mutations have factor VII levels between 20% and 60%, whereas homozygous levels tend to be less than 10%, but these levels can vary based on the specific mutation . The mosIn other bleeding disorders, heterozygotes usually do not have severe bleeding because the normal allele of the factor gene contributes 50% of the normal levels which is sufficient for hemostasis. However, the clinical severity of F7D correlates poorly with factor VII levels which can range from 50% to 200%, and the role of factor replacement therapy is not clearly defined. The half-life of factor VII is 4-6 hours with a targeted level of 15-20% required at minimum to prevent spontaneous bleeding [According to a systematic review of the literature on the management of pregnancies complicated by F7D, the hemorrhage rates are equivalent in patients with and without hemostatic prophylaxis. The recommendation is to consider prophylaxis use of recombinant factor VII or factor VII concentrate as part of an individualized discussion in the context of prior bleeding complications and mode of delivery . To improve patient outcomes, hemostatic agents should be available in case of hemorrhage or surgical intervention . PossiblAs mentioned prior, pregnancy itself is a prothrombotic state due to the numerous metabolic, immunological, and hemostatic changes that take place in the body. In pregnancy, plasma volume can expand up to 40%, with RBC expanding only 25%, and platelets also experience a relative decrease due to dilution and sequestration by the placenta. These effects are offset by an increase in coagulation factors. However, in some coagulation disorders, the deficiency of certain factors can still place pregnant patients at risk, especially during the postpartum period. Von Willebrand disease (VWD) is one of the most common bleeding disorders found in the general population and affects upwards of 50,000 deliveries annually. Patients with type 1 VWD disease attain normal factor levels as pregnancy progresses, while those with type 2 and 3 VWD disease are more at risk for hemorrhage, especially during the first 24 hours postpartum. Patients with hemophilia A and B  are usually asymptomatic carriers; however, a large proportion still experiences coagulation factor levels far below normal during pregnancy. While factor VIII levels increase throughout the pregnancy gestation, factor IX levels do not increase as much, which makes supplementation to normal non-pregnancy levels a necessity if the patient has a history of bleeding or low factor levels . ManagemThe management of pregnant patients with F7D remains a challenge to clinicians in balancing the risk of thrombosis and hemorrhage. The level of factor VII does not always correlate with clinical severity and there are no concrete guidelines for the indications or dosing criteria for factor VII replacement therapy. Taken together, management of the delivery for pregnant patients with F7D should be addressed on a case-by-case basis at centers where a multidisciplinary team with expertise is readily available. Managing this patient’s bleeding risk depends on bleeding severity, clinical history, the initial level of factor VII, and the availability of factor replacement products, ideally recombinant activated factor VII. Patients wishing for further pregnancies should continue to require regular visits with outpatient hematology to monitor factor VII levels, as having F7D could increase the chances of having recurrent miscarriages. Regular follow-up with therapy and counseling services, including mental health and reproductive health, and OBGYN would also benefit and empower the patient for future fertility plans.


The vaginal samples of the healthy participants were collected throughout pregnancy, that is, during the first, second, and third trimesters. The V3–V4 region of 16S rRNA was used to analyze the vaginal microbiome, and the bioinformatic analysis was performed using QIIME Alpha and Beta diversity analysis.Alloscardovia was only found in participants who conceived after IVF, and the percentage of Alloscardovia in viginal samples of normal delivery group is much higher than the samples from preterm delivery group.Vobrio specifically colonized in vagina of pregnant woman in AFT group (those who conceived after IVF (A), first trimester (F), and delivered at term (T)) and Sporosarcina was detected only in women with AFT and AST (those who conceived after IVF (A), second trimester (S), and delivered at term (T)). These data indicates that Alloscardovia, Vobrio and Sporosarcina have great potential in predicting pregnancy outcomes who pregnanted by vitro fertilizationEither IVF group or Natural conception group, bacterial community diversities and total species number of vagnal samples from who delivered at term were significantly higher than those who delivered before term. Low abundance of vaginal bacteria indicates an increased risk of preterm delivery. Further, more abundant vaginal bacteria was found in first trimesters instead of the next two trimesters. Vignal samples collected during first trimester showed richer differences and more predictive value for pregnancy outcoes. In addition, the diversity of the vaginal bacterial community decreased as the gestational age increased, in all samples. Oceanobacillus might act as a positive biomarker, whereas Sulfurospirillum and Propionispira may act as negative biomarkers for the risk of preterm birth.Vaginal microbiota were more stable in women who conceived naturally and those who carried pregnancy to term.  However, IVF . All operations and evaluations were performed by the same doctor. In total, 19 women had conceived after IVF after a long protocol of conventional solutions of fresh-cycle IVF and conventional luteal support after 1 week; 6 of these delivered before term, whereas 13 delivered at term. Six pregnant women had visited the obstetrics and gynecology clinic for a routine pregnancy test during the same period; three of these women delivered before term, whereas three delivered at term.The inclusion criteria for this study were as follows: (1) No symptoms of vaginitis, single live births, and primipara; (2) abstinence from sex for 2 weeks, no antibiotic use, no medication history related to the vulva and vagina for 1 month prior to the study, and no history of systemic use of hormone drugs and immunosuppressants; (3) no history of pregnancy complications such as vaginitis, gestational hypertension disease, gestational diabetes mellitus, or pregnancy with abnormal thyroid function; and (4) no history of smoking, drinking, or drug consumption.Vaginal swab specimens were collected throughout the prenatal check-ups of the healthy participants in all three trimesters of pregnancy. The swabs were obtained at routine prenatal visits, and by rotating a sterilized swab five times along the vaginal lumen in a circular motion. Speculum was not used. No occurrences of premature rupture of membranes was observed, and no participant was administered intravenous antibiotics during delivery. The participants were divided into those who conceived after IVF and those who conceived naturally (controls), and according to the mode of delivery, as full term (≥37 weeks) and preterm (<37 weeks). The collected swabs were placed into sterile tubes by trained research staff and stored at −80°C for DNA extraction.The vaginal swabs were collected at least in triplicate at three time points: first trimester , second trimester , and third trimester  of pregnancy. The presence of specific vaginal bacterial communities in any single gestational period in participants who delivered before term  was compared with that in participants who delivered at term .2.2The standard long IVF regimen was used. A gonadotropin-releasing hormone (GnRH) agonist regimen was the main protocol used in this study, and all patients underwent embryo transfer in fresh cycles. Controlled ovarian hyper-stimulation, oocyte retrieval, and embryo transfer were carried out. Participants were treated with downregulation from the mid-luteal phase of the previous cycle. When the pituitary reached desensitization, recombinant FSH was begun at 150–225 IU/day. Human chorionic gonadotropin (hCG) was given  once two or more follicles had reached a size of 18 mm. Oocytes were extracted 34–36 h after the hCG trigger, and this was followed by intracytoplasmic sperm injection (ICSI). Participants were injected progesterone  at 40 mg/day 48 h after oocytes fertilization. The progesterone supplementation was continued until 10 weeks of gestation after pregnancy was achieved.2.3® High-Fidelity PCR Master Mix (New England Biolabs) was used for PCR amplification according to the selection of the sequencing region.Genomic DNA was extracted using cetyltrimethylammonium bromide; the concentration and purity of DNA were detected by 1% agarose gel electrophoresis. The appropriate amount of sample was placed into the tube and diluted with sterile water to 1 ng/μL. High-throughput sequencing technology was used to sequence the V3–V4 region of the 16S rRNA gene, which was amplified used a specific primer with the barcode 314F-806R (V3V4 primers: 314F-5′ CCTAYGGGRBGCASCAG 806R5′ GGACTACNNGGGTATCTAAT) Phusion2.4PCR products were mixed with the same volume of 1× loading buffer and detected on 2% agarose gel electrophoresis. Sample strips of 400–450 bp were chosen for further experiments. The PCR products were purified using Qiagen Gel Extraction Kit .2.5® DNA PCR-Free Sample Preparation Kit  was used to generate sequencing libraries, and their quality was analyzed using the Qubit@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system. The library was sequenced on Illumina HiSeq2500 platform, and finally, 250-bp paired-end reads were generated.TruSeq2.6The reads were merged by FLASH to obtain raw tags (V1.2.7) , and qua2.7t-test and mapping between groups was done using R software (Version 2.15.3).Alpha diversity was exhibited by six indices, namely Observed-species, Chao1, Shannon, Simpson, ACE, and Good’s Coverage, which were calculated with QIIME (Version 1.7.0) and displayed using R software (Version 2.15.3). Beta diversity was calculated by QIIME software (Version 1.7.0) and R software (Version 2.15.3). Species analysis with significant differences between groups was performed using Student’s 2.8We Uploaded the raw 16S rRNA gene sequencing data to the National Center for Biotechnology Information (accession no. PRJNA728871).33.1via IVF(A)was 31.42 ± 10.23 years, and that of those who conceived naturally (B) was 31.50 ± 4.52 years (P > 0.05). The mean BMI of the participants in Group A was 26.57 ± 2.03 kg/m2 and that of participants in Group B was 27.48 ± 0.95 kg/m2 (P > 0.05); The mean fetal weight in Group A was 3157.89 ± 628.55 g and that in Group B was 2991.67 ± 452.12 g (P > 0.05).The details of the participants are stated in via IVF  and those who conceived naturally  are stated in The details of the specimens collected from the participants who conceived 16S rRNA of each sample was analyzed using high-throughput sequencing technology. The rarefaction curves tended to approach saturation in all samples except for sample BFT , first trimester (F), and delivered at term (T))Figure 13.2via IVF (A) or naturally (B), the number of species and diversities in the bacterial community in those who delivered at term (T) were significantly higher than those in women who delivered before term (P) throughout pregnancy , regardless of the pregnancy pattern. The numbers of the observed species were as follows: AFT > AFP, AST > ASP, ATT > ATP, BFT > BFP, BST > BSP, and BTT > BTP  and before term (P), the vaginal bacterial communities in the first trimester were higher than those in the other two trimesters. This indicated that the first trimester was a critical period during pregnancy, and the diversity of vaginal bacterial communities decreased with increasing gestational age in all samples Figure 2The results indicated that conception after IVF could be associated with significantly decreased number of VMB in the first trimester, and that reduced microbial diversity in the vagina could be associated with an increased risk of preterm birth.3.3Lactobacillus was dominant in all the samples (>93%), which was consistent with previous reports  in all the samples, followed by Proteobacteria (0.133%), Actinobacteria (0.052%), and Bacteroidetes (0.050%). Actinobacteria appeared only in groups AT and AP (mostly in group AT), and Sulfurospirillum was found only in group BT. Vibrio was found only in group AT  and those who conceived naturally (B), and between women who delivered before term (P) and those who delivered at term (T) Figure 43.5t-test was performed to determine the significant difference between the bacterial species in all the groups (P < 0.05), and the results showed significant differences between groups AP and AT. Clostridia were a significant differential species and were dominant in group AT; between groups BP and BT, unidentified Actinobacteria were a significant differential species and were dominant in group BT; between groups AT and BT, Lactobacillus equicursoris was a significant differential species and was dominant in group AT , indicating that early pregnancy is a significant period for the vaginal bacterial community, and that the fluctuation of vaginal bacteria in the first trimester may be a biomarker for preterm birth.We found that the community richness and diversity of VMB was lesser in women who conceived after IVF than in those who conceived naturally throughout pregnancy, regardless of the delivery pattern, suggesting that the VMB were more stable in naturally pregnant women than in those who were pregnant after IVF. The number of observed species was lesser in women who conceived Lactobacillus was predominant in the vagina of naturally pregnant women , Vibrio was found only in group AT, and Sulfurospirillum was found only in group BT. Alloscardovia was specifically detected in women who conceived via IVF and could act as a positive biomarker, whereas Vibrio and Sporosarcina could act as negative biomarkers for the risk of preterm birth. Oceanobacillus might act as a positive biomarker, whereas Sulfurospirillum and Propionispira might act as negative biomarkers for the risk of preterm birth in women who conceive naturally.Consistent with this observation, our data also showed that this genus was dominant in women who were pregnant after IVF. To determine the specific taxa in women who were pregnant after IVF, the details of vaginal bacteria were analyzed; we observed that Oceanobacillus, a member of the Baciliaceae, was isolated from deep-sea sediments by Lu et al.  using O.Kumar M and Hočevar K , 53 idenThe main reason for the different research results may be that all our samples were after IVF and some vaginal operations were performed before pregnancy.So far the characteristics of vaginal Microecology in pregnant women after IVF have not been described.19 women had conceived after IVF after a long protocol of conventional solutions of fresh-cycle IVF, our sample pregnant women are completely infertile due to male factors, avoiding the changes of vaginal microecology caused by changes in drug and hormone levels. However, before becoming pregnancy, these pregnant women will have repeated vaginal ultrasonography, vaginal flushing and transvaginal fornix oocyte collection, which will destroy the integrity of vaginal flora, These reasons may be the reasons for the differences in vaginal flora samples. Because our samples are all male factors, this also avoids the impact of changes in maternal hormone levels on vaginal flora.Before pregnancy, vaginal flora structure was destroyed after vaginal flushing. So after pregnancy, vaginal flora structure also means reestablishment. For this reason perhaps the number of species and diversities in the bacterial community in those who delivered at before term throughout pregnancy were lower.These results are currently observed in our research. The specific mechanism is not very clear. We will study it in the next research.Second, due to the small number of pregnant women with simple male factor and insufficient sample size, the results may also be different. We will increase sample size in the next research.We recruited 19 volunteers in IVF group and 6 volunteers in Natural conception group and collected vaginal samples from each volunteer in three periods. These samples are very precious and can effectively remind us of the relationship between vaginal bacteria and pregnancy outcomes. In our results, we found vaginal samples collected during first trimester showed richer differences and more predictive value for pregnancy outcomes. In addition, these data indicates that Alloscardovia, Vobrio and Sporosarcina have great potential in predicting pregnancy outcomes who pregnant by vitro fertilization. While Oceanobacillus, Sulfurospirillum and Propionispira have great potential in predicting the risk of preterm birth in women who conceive naturally.On the other hand, the sample size in our research is not enough to conclude these bacteria strains we suggested could be biomarker for preterm birth. And the mechanism of these bacteria strains on preterm birth is unclear. But our data is a powerful indicator.Although the exact causal relationship remains to be determined, our results confirm an association between some bacteria and preterm birth. However, the richness, diversity, and stability of the microbiome may be important during pregnancy. Our finding is consistent with previous studies , 55.via IVF and those who deliver before term show lesser richness, diversity, and stability of VMB. Therefore, standard protocols should be established and used to support a shift of VMB.In summary, our study showed that compared with naturally pregnant women, women who conceive https://www.ncbi.nlm.nih.gov/, PRJNA728871.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Dalian Women’s and Children’s Medical Center . The patients/participants provided their written informed consent to participate in this study.YT: responsible for the experimental concept, data collection, drafting papers; QS: responsible for the data collection, drafting papers; XS: Responsible for the important revision of the thesis; ZW: Responsible for the important revision of the thesis and for the important revision of the thesis. All authors contributed to the article and approved the submitted version.