CoV2), which should play a key role in a new drug development and in the interaction with RNA and other proteins are summarized. The structural feature of NCoV2 is first described. Next, simulation studies on the interaction between potential drug molecules and NCoV2 are summarized, which show the importance of molecular flexibility. Then, liquid-liquid phase separation phenomenon involving NCoV2, which has recently been reported, is described. This phenomenon also suggests the importance of molecular flexibility of NCoV2. Finally, a promising method using neutron scattering to characterize the structure and structural fluctuation of the droplets, which are formed through this phenomenon, is presented.Nucleocapsid protein is one of the essential proteins for viral replication including the coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) pneumonia leading to the ongoing pandemic. Whereas this protein has emerged as a potential drug target, its physicochemical properties and the molecular mechanism of how this protein is involved in viral replication remain unclear. In this review, structural and dynamical aspects of the SARS-CoV-2 nucleocapsid protein (NCoV2) is one of the abundant proteins and shows multiple functions including packaging the RNA genome during the infection cycle. It has also emerged as a potential drug target. In this review, the current status of the research of NCoV2 is described in terms of molecular structure and dynamics. NCoV2 consists of two domains, i.e., the N-terminal domain (NTD) and the C-terminal domain (CTD) with a disordered region between them. Recent simulation studies have identified several potential drugs that can bind to NTD or CTD with high affinity. Moreover, it was shown that the degree of flexibility in the disordered region has a large effect on drug binding rate, suggesting the importance of molecular flexibility for the NCoV2 function. Molecular flexibility has also been shown to be integral to the formation of droplets, where NCoV2, RNA and/or other viral proteins gather through liquid-liquid phase separation and considered important for viral replication. Finally, as one of the future research directions, a strategy for obtaining the structural and dynamical information on the proteins contained in droplets is presented.The latest coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) pneumonia leading to the pandemic, contains 29 proteins. Among them, nucleocapsid protein (N CoV2).A new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the cataclysmic pandemic through coronavirus disease 2019 (COVID-2019) pneumonia. SARS-CoV-2 is a member of β-coronavirus family that includes well-known SARS-CoV and MERS-CoV , and 4 mCoV2, which is composed of 419 residues in total, is shown in CoV2 consists of two domains, the N-terminal domain  and the C-terminal domain . The NTD is responsible for RNA-binding and the CTD for dimerization and oligomerization  and the C-tail (365–419), respectively. The atomic structures of the NTD have recently been solved by X-ray crystallography at 2.7 Å resolution /[polyU]) of 0.6 (wt/wt), heterotypic droplets (peptides-RNA droplets) were transformed into homotypic droplets (RNA-RNA droplets). Another study has shown that formation and dissolution of the peptide-polyU droplets are regulated by the phosphorylation state of the peptides [CoV2 droplets, indicating that interactions occurring at the amino acid level are prerequisites for droplet formation.In addition to proteins, small peptides are also known to show phase separation with RNA through electrostatic interactions ,74,75. INA ratio . In thisCoV2 droplets are dynamic objects: when NCoV2 or RNA molecules are added to the preformed NCoV2/RNA droplets, both types of molecules are incorporated into the droplets and simultaneously the NCoV2 and RNA molecules that constituted the preformed droplets diffuse out of the droplets, suggesting that both NCoV2 and RNA are exchangeable between the dense and dilute phases [CoV2 and RNA are crucial for dynamically maintaining the droplets. Moreover, when the complex of RdRp and RNA is added to the NCoV2/RNA droplets, the entire RdRp-RNA complex is also incorporated into the droplets [CoV2/RNA and G3BP1, there are three populations of the NCoV2 molecules that diffuse at different mobility [CoV2 molecules in the droplets might play an important role in the transcription and replication cycle. Since the diffusion of the whole protein molecules inside the droplets is largely affected by the strength of interaction between NCoV2 and other proteins/RNA, it would be of great interest to understand the relevant physical properties of NCoV2 such as side-chain fluctuations, which directly determine the rate and the manner of interaction with their binding partners.Fluorescence measurements using Alexa fluorophores have shown that the Ne phases . This aldroplets , suggestmobility . These rCoV2, RNA, other viral proteins and a stress granule protein are concentrated into a different phase from its surroundings suggest that the NCoV2 droplet formed through LLPS is the main arena where molecular events critical for the virus replication take place. It is thus indispensable to reveal the internal organization of NCoV2 droplets, especially the structure of NCoV2 in droplets formed with various binding partners to develop a new intervention technique targeting NCoV2 as well as to understand the molecular mechanism of biological functions of NCoV2 in detail. Furthermore, the molecules within the droplets are not frozen to the spot but fluctuate dynamically around their average conformation under the influence of thermal energy. The nature of these structural fluctuations is closely related to the interaction, the stability and the function of the proteins. Thus, unraveling the structure and structural fluctuation of NCoV2 in the droplets formed in various conditions is essential and the research in this direction would occupy an important position in this research field.The recent findings that NCoV2 in droplets would be small-angle neutron scattering (SANS). This technique has been used to characterize the structure of viral proteins in isolated state [CoV2 droplets. In neutron scattering, the strength of scattering (coherent scattering length) depends on the type of atoms bombarded by neutron as shown in 2O in the solvent. As the D2O content in the solvent increases, the labile hydrogen atoms in the solutes are exchanged for deuterium atoms, which modifies the SLD of the solutes. The SLD of perdeuterated proteins , meaning that the scattering contrast of the H-protein is zero. In this condition, the contribution of the H-protein to the scattering signal is effectively zero and hence it is “invisible” to neutron. Even though techniques to perdeuterate or partially deuterate RNA molecules by chemical synthesis, in vitro transcription and in vivo production have been established for NMR studies [A powerful method to characterize the molecular structure of Ned state ,77 and ied state ,79,80. Hed state ,82,83,84 studies ,89, prod studies . TherefoCoV2 structure in droplets. In the case where droplets are formed by NCoV2, RNA and another protein , (2) those formed by H-NCoV2, H-RNA and the perdeuterated another protein (D-AP) and (3) those formed by the perdeuterated NCoV2 (D-NCoV2), H-RNA and D-AP. The SANS measurements in these samples in the ~65% D2O solvent, the SLD of which matches that of H-RNA, provide three kinds of scattering data where each protein contribution is different. From these data, the scattering data arising from each protein can be extracted, leading to the elucidation of its conformation together with how each protein is arranged inside the droplets. protein c, three 2O buffer can minimize the solvent contribution to the iNS spectra. Since the hydrogen atoms are quasi-uniformly distributed throughout the molecules in the biological systems, the dynamical information averaged over the whole molecule is obtained. In the case of proteins, the observed motions reflect the fluctuations of amino acid side chains and backbones because hydrogen atoms are bound to these chemical groups. The schematic illustration of the dynamical analysis of the NCoV2 droplets is depicted in CoV2, RNA and another protein, two types of samples are required: (1) droplets formed by H-NCoV2, H-RNA and D-AP, (2) those formed by D-NCoV2, H-RNA and D-AP. By subtracting the iNS spectra of the latter from those of the former measured in 100% D2O, the remaining spectra arise from the structural fluctuation of the NCoV2 molecules, thereby providing dynamical information on NCoV2 inside the droplets. Instead, the dynamics of another protein in the droplets can be extracted in a similar manner.The structural fluctuation of the molecules in the droplets are obtained by incoherent neutron scattering (iNS). This technique measures the intensity of neutron scattered by the samples as a function of the momentum and energy change of the neutron, from which the amplitude, the frequency and their distribution of atomic motions at pico- to nanosecond timescale at ångström length scale are estimated . The valD) of the identical NCoV2 molecule and the distance between domains of different NCoV2 molecules (RM) can be determined from structural analysis. This means that not only the conformation of NCoV2 in droplets but also the arrangements of the NCoV2 molecules in droplets are revealed. From the iNS measurements, efficient diffusion coefficients (Deff), which include translational and rotational diffusion, of the whole NCoV2 molecule and/or domains will be obtained. Furthermore, as shown in the lower panel of CoV2 in droplets with that in an isolated state (CoV2 in droplets. It is likely that the unique structure and arrangements of proteins inside the droplets form a field where RdRp molecules bind to RNA more easily than outside the droplets, promoting the viral genome transcription. The unique mode of protein motions inside the droplets would be the determinants of interactions between other molecules inside the droplets, affecting the efficiency of genome transcription. All the above structural and dynamical information will thus be useful not only for understanding the molecular mechanism of the virus replication/infection cycle but also developing medical intervention techniques based on the structure and dynamics of proteins. Moreover, since the time- and space-scales of iNS are comparable to those of MD simulation, dynamics parameters obtained from iNS would serve as a fundamental information in in silico drug development targeting the droplets in the future.That way, the structure and structural fluctuation of the proteins contained in the droplets, which play a crucial role in virus replication, are characterized. For example, following parameters characterizing the internal organization of the droplets are found: as shown in ed state b and in ed state c, structCoV2 plays a critical role in drug-binding, RNA-binding and formation of the droplets. Knowledge of the structure and dynamics of the NCoV2 molecules in isolated states in solution and in a variety of droplets would advance our understanding of the architecture and the physical properties of the droplets, thereby shedding light on the molecular mechanism of the viral infection and replication cycle. Furthermore, all this information would eventually be beneficial to the development of new drug molecules targeting NCoV2, of improved diagnostic techniques, of a new type of treatment for the COVID-19 pneumonia, and of a new combat strategy for a future pandemic.In conclusion, the molecular flexibility as well as conformations of N


Lastly, an eight-day pozzolanicity analysis (PA) was carried out to determine the pozzolanic reactivity of the samples. The results obtained by XRD, XRF and SEM established that the researched zeolite samples have two main zeolitic phases: mordenite and clinoptilolite. Altered volcanic glass, quartz and smectite (montmorillonite) are the secondary phases. The results of the chemical quality analysis (CAQ) showed that the samples contain a considerable amount of reactive SiO2 and reactive CaO, as well as a low content of insoluble residue, which reinforces their properties as pozzolans. The results of the pozzolanicity analysis (PA) concluded that the analysed samples actively react with Ca(OH)2 after eight days. Based on all the results mentioned above, it is established that both mordenite and clinoptilolite behave like pozzolans and can be recommended for the manufacture of pozzolanic cements, which have more effective properties than Portland cement, in terms of physical, chemical and mechanical strength, low heat of hydration, resistance to sulphates, low CO2 emissions to the atmosphere and negligible impacts on the environment.This work describes the newly discovered zeolites in the eastern region of Cuba. In the researched area, there have been no previous studies of natural zeolite exploration. Therefore, the results shown here are new. The main object of this research is to analyse five samples of zeolites and demonstrate their pozzolanic capacity and the possibility of their usage in the industrial manufacturing of pozzolanic cements. The study of the samples was performed by X-ray diffraction (XRD), X-ray fluorescence (XRF) and scanning electron microscopy (SEM). A chemical analysis (CAQ) to determine the quality of the samples as pozzolans was performed, by determining the total SiO Several magmatic cycles formed the volcanic arc of Cuba during the Cretaceous. The erosive process created large deposits of volcano-sedimentary materials in the deposition basins; many of these materials were enriched with volcanic glass that was gradually altered by meteoric waters and hydrothermal processes, forming large deposits of zeolites in many places in Cuba. This paper presents the results of the research on the characterisation of new indications of natural zeolites of the eastern part of the Republic of Cuba, in the north-west of Holguín . KnowledZeolites were first described in the province of Holguín in 1971 by geologists of the Institute of Geology and Paleontology (IGP) of La Habana (Cuba) . SubsequCurrently, zeolites are widely used in various fields of the Cuban economy. The clinoptilolite is used to promote growth in plants and is a very valuable fertiliser . It is uThe present work had two main phases. In the first phase, a mineralogical, petrological and chemical study of zeolites found in an unexplored area in the eastern region of Cuba was carried out; in the second phase, their technological qualities were determined in order to establish their properties as pozzolans. The studies carried out in both phases considered three fundamental questions: the geological nature of the deposit, the pozzolanic properties of these zeolites and the possibility of their use as a raw material in the manufacture of pozzolanic cements.The area of study is within the Loma Blanca Formation from the Lower Cretaceous Aptian to the Upper Cretaceous Campanian . It is dThe results obtained by XRD established that the researched zeolite samples have two main zeolitic phases, mordenite and clinoptilolite, which is evident in the X-ray diffraction patterns presented in The peak of maximum intensity (Imax = 100%) of the mordenite is located at 2θ = 26.65   is located at the angular position 2θ = 9.88 and is overlapping with a mordenite peak. A second peak of clinoptilolite of appreciable intensity is seen in 2θ = 11.19, with an intensity equal to 40%. Other peaks with less intensity appear at angular positions 13.06 to 32.01, with intensities varying between 14% and 48.2%.Above 2θ = 45.0, the peaks of clinoptilolite are masked by mordenite, which is interpreted as an isomorphic substitution between the two minerals, possibly caused by a variation in the alkalinity/acidity of the geological environment ,21,24. TQuartz has its peak of maximum intensity (Imax = 100%) shifted towards the angular position 2θ = 26.54. Peaks with medium intensity are observed distributed at positions 2θ from 36.45 to 59.87. Quartz peaks with even lower intensity have not been taken into consideration in the discussion, although they can be seen in 2O and Na2O have higher values than MgO; this fact explains why the mordenite–clinoptilolite phase was developed more than the smectite phase (montmorillonite). A similar approach has been formulated in the last decade by Costafreda  was performed through the following expression [−]: is the concentration in hydroxyl ions (mmol/L); V3: volume of the hydrochloric acid solution (0.1 mol/L); f2: the factor of the hydrochloric acid solution (0.1 mol/L).The procedures for the chemical test of pozzolanicity were adapted to the UNE-EN 196-5:2011 standard . First, pression .(8)OH−=4: the volume of EDTA solution used in the titration; f1: the factor of the EDTA solution.The concentration of calcium oxide (CaO) was calculated through the following equation .(9)CaO=According to the UNE-EN 196-5:2011 standard , the mix2 and Al2O3, which indicates that the zeolites analysed are undoubtedly good-quality pozzolans for use in the manufacture of pozzolanic cements, without negative impacts on the environment. These cements have the property of resisting the attack of seawater and sulphates, which is beneficial for the Island of Cuba, where the main cities and tourist infrastructures are concentrated on the coast. Lastly, the manufacture of pozzolanic cements is low in cost, which could have a positive impact on the economy of this country, principally in the construction of cheap housing for the population, which is often affected by hurricanes and earthquakes.The results presented and discussed demonstrate a close genetic and spatial relationship between mordenite and clinoptilolite. Both minerals have been formed as secondary species through a process of devitrification and hydrothermal alteration of the volcanic glass. The chemical composition shows appreciable values of SiO


To study the state of immunity in pregnancies associated with urogenital infection and complicated by intrauterine infection.nd and 3rd trimesters of gestation. Depending on the outcome of each pregnancy on the basis of the follow-up of newborns, performed on the first day after birth, the patients were retrospectively divided into two groups. The study group included 93 (37.2%) pregnant women who developed intrauterine infection. The comparison group (n=157 (62.8%)) comprised pregnant-carriers of perinatally significant infection who gave birth to conditionally healthy children. The control group consisted of 50 healthy women with a physiological pregnancy.The comparative study involved the examination of 250 pregnant women with urogenital infection and ultrasonographic signs of intrauterine infection and their newborns in order to assess the state of cellular and humoral immunity components and nonspecific resistance. A direct prospective examination of pregnant women was carried out in the 2In the gestation period under investigation, the development of intrauterine infection in pregnant women with urogenital infections was found to be associated with a deficiency of T-helpers / inducers, an increase in thymus-dependent lymphocyte killer activity, a high content of IL- 1β, TNF-α in the systemic circulation, and a decrease in the level of IL- 10 secondary to the oppression of the effector link of phagocytic neutrophils of peripheral blood.An increased concentration of systemic proinflammatory cytokines IL-1β, IL-6 and TNFα with a simultaneous decrease in the IL-10 content and suppression of the killing activity of peripheral blood phagocytes reflects the presence of an active inflammatory process in the mother-placenta-fetus system and can be one of the factors affecting the development of intrauterine infection in pregnancy, complicated by urogenital infection. An increase in the incidence of intrauterine infection (IUI) is the most urgent problem for obstetricians, as it is one of the leading causes of morbidity and mortality in peri- and neonatal periods of fetal and neonatal development , 2.The iIn Ukraine the frequency of intrauterine infections (IUI) ranges from 6 to 53%, reaching 70% among preterm infants. In the structure of perinatal mortality, the proportion of IUI ranges from 2 to 65.6% , 5, 6, 7It is known that intrauterine infection as a result of infectious matter invasion into the fetus does not always develop into a fetal infection, i.e. one in which the introduction of an infection by means of the penetration of a pathogen from an infected mother is expressed by a number of clinical manifestations in the early neonatal period .The lattNumerous studies have shown that infections are detected in almost every second birth , 15, 16.The leading role in the pathogenesis of pathological conditions developing in the perinatal period is played by sexually transmitted infections , 18, 19.Thus, changes in the immune system during gestation may be due to an increased risk of infection.Therefore, it is relevant to study the issues related to the search for markers for assessing the risk of intrauterine infection and its implementation .The purpose of the study: to assess the features of the state of immunity in pregnancies associated with urogenital infection and complicated by intrauterine infection.The study involved 250 pregnant women with urogenital infectious pathology and the presence of reliable signs of intrauterine infection.The gestation period was 28-37 weeks and was determined by the comparison of clinical and medical history data and ultrasonic fetometry findings.The inclusion criteria were as follows: echographic IUI signs, singleton progressive unstimulated pregnancy, patient’s informed consent for the use of biological material for scientific purposes. Exclusion criteria were: multiple pregnancy, pregnancy with rhesus-sensitization, severe somatic pathology and chronic diseases in the decompensation stage , previous stimulation of ovulation, IVF, chromosomal abnormalities and congenital malformations of the fetus.The distribution of patients by groups was carried out on the basis of retrospective analysis of immunity indices under investigation during the gestational period and the results of the follow-up examination of newborns performed in the first 72 hours after birth.The study group included 93 mothers, whose newborns had clinical signs of intrauterine infection.The comparison group comprised 157 patients with perinatally significant urogenital infections of di&erent origin, who gave birth to conditionally healthy children without signs of infection developing.The control group consisted of 50 conditionally healthy pregnant women with a physiological pregnancy at a similar gestational age in the absence of bacterial and viral infection on the basis of serological and microbiological research methods, whose infants were born without deviations in physical development and functional status. All the pregnant women gave natural birth at term (39-41 weeks).Cl. trachomatis, Ureaplasma urealiticum, Mycoplasma hominis, Mycoplasma genitalium, Nisseria gonorrheae, T. vaginalis, as well as herpes  and cytomegaly (CMV) viruses.Infectious status was determined by etiological interpretation using polymerase chain reaction (PCR) and enzyme immunoassay (ELISA) to detect The state of cellular immunity components was identified by the phenotypic characteristics of peripheral blood lymphocyte populations and subpopulations, activation markers (HLA-DR) and receptors for IL-1β, IL-6, IL-10, TNF-α cytokines.The subpopulation composition of lymphocytes was determined on a FACSCalibur (USA) flow cytometer (CellQuest Pro software) using standard protocols. TNF-α was detected in blood serum by enzyme immunoassay with the use of reagent kits manufactured by ZAO Vector-Best  according to the attached instructions; TNF-α concentration was expressed in pg / ml. IL1-β, IL-6, IL-10 in blood serum was determined by solid-phase enzyme-linked immunosorbent assay using sets produced by ZAO Vector-Best  according to the manufacturer’s instructions. Optical density of the solutions was recorded on a microplate reader, and the concentration was expressed in pg/ml.The state of humoral immunity components was assessed by the content of the immunoglobulins of the main classes, particularly G, A, M and circulating immune complexes in blood serum.The concentration of G, A, M immunoglobulins in blood serum of the pregnant women examined was determined by simple radial immunodi&usion , based oNonspecific resistance of the body was determined by phagocytic activity of peripheral blood neutrophils by the method of completed phagocytosis  using opThe majority of the women studied, i.e. 150 (60%) out of 250 had mixed viral-bacterial associations (VBAs), while virus-only infections were detected in only 50 (20%) and bacterial-only infections in 50 (20%) of the pregnant women. All the patients examined were comparable in age, social status, past gynecological and extragenital diseases.Statistical analysis was carried out with the help of the “STATISTICA 6.1” application software (Russified version). Statistical significance of the intergroup di&erences was checked by the nonparametric Mann-Whitney test (MWT).The assessment of the results obtained .The depElevated levels of lymphocytes with CD3+ CD16 / CD56+ (NK tot) phenotype in the group of women with IUI development indicated an increase in the killer activity of lymphoid cells.It should be noted that the relative content of B-lymphocytes (CD19+) in pregnant women with the development of maternal and fetal infections did not significantly differ from the control indices (p>0.05).Assessment of the state of the cytokine system in both the study group and the comparison group without the development of intrauterine infection showed a significant increase in pro-inflammatory cytokines IL-1β, IL-6 and TNFα.IL-1β concentration in the study group was almost 5 times higher than in the control group and 3 times that in the comparison group. A high IL-1β level in the systemic blood flow of patients in the study group may indicate activation of phagocytosis and cytolysis .IL-6 content in peripheral blood serum of the study group was 4 times higher than in the control group and almost 2-fold that in the comparison group. Expression of IL-6 in biological fluids is always accompanied by great variability and depends on many factors a&ecting the intrauterine state of the fetus, including mixed infection . In addiTNF-α content was almost 3 times higher than in the control group and 2 times that in the comparison group. TNF-α is an important cytokine involved in the development of inflammatory response, stimulating the activity of natural killers and cytotoxic lymphocytes, contributing to the development of a response to various infectious agents .The results of IL-10 content assessment revealed an almost 4-fold and 2-fold decrease in its concentration in the main group in relation to the comparison and control group, respectively. A reduction of the content of this anti-inflammatory cytokine in the development of intrauterine infection indicates an impairment of adaptation of immunoregulatory mechanisms in IUI development .Immunoglobulins play an important role as mediators in the cascade development of immune response and can partially determine the effectiveness of the final, effector responses of cellular immunity in inactivating and eliminating bacterial, viral and fungal antigens.Assessment of humoral immunity  indices showed a significantly higher content of IgM, a primary immune response mediator, in the pregnant women of the study group, as compared to the control and comparison group, which may indicate the activation of antibody-dependent cellular cytotoxicity in an active course of infection  was within the normal range, but it was in the upper limit of its reference values, which corresponded to the literature data on the increase of this index during the entire gestation period, reaching a maximum in the 2imesters . IgG conThe content of circulating immune complexes (CIC) is an integral indicator of the functional activity of humoral immunity. The study showed that CIC concentration in the serum of the patients of the study group was significantly higher (p <0.05) than in the control group.CIC formation is an obligatory component of normal immune response, one of the most important humoral mechanisms of antigen elimination and a universal defense mechanism aimed at maintaining the internal environment of the body. Furthermore, CIC is able to interact with most immunocompetent cells and cells of the monocyte-phagocytic system, which is expressed in the production and secretion of biologically active substances.Neutrophils are the main effector cells controlling infectious agents. Assessment of the functional reserve of phagocytizing neutrophils of peripheral blood of the pregnant women under investigation showed that the absorption capacity of phagocytes in all the groups of the patients examined was practically on the same level as their bactericidal activity (BAN). However, the latter was accompanied by a significant (p<0.05) decrease in the digesting capacity of phagocytizing neutrophils in the blood of the pregnant women who developed intrauterine infection – the index of phagocytosis completeness was 0.89 st.un. against 1.15 and 0.94 st.un. in the control and comparison group, respectively (The physiological course of pregnancy, in the absence of an infectious factor, as a result of an increase in the total number of neutrophils in blood enhancing their ability to capture opsonized antigenic material, and increasing their bactericidal activity, is characterized by the development of a powerful and mobile neutrophil reserve to perform full phagocytic function, indicating that CIC content is under effective control, which is confirmed by our study. In the development of intrauterine infection, high CIC concentration in blood serum of the patients of the study group is associated with insufficiency of their clearance from the blood flow [37].Thus, our analysis of the data obtained allows us to conclude that the state of immunity in pregnant women with urogenital infections and IUI development is characterized by a deficiency of T-helper/inducer lymphocytes, an increase in CD3+ CD16+ / CD56+ cells, an imbalance of anti- and pro-inflammatory cytokines, with a prevalence of the latter. The development of the inflammatory process in the mother-placenta-fetus system, which is confirmed by the high content of IL-1β and TNF-α in the systemic circulation, is accompanied by the activation of the humoral immune link, which is manifested by an increase in the level of circulating immune complexes. This fact may indicate that CIC accumulation in peripheral blood during the implementation of intrauterine fetal infection is not so much triggered by their excessive formation in response to antigenic stimulation, but rather by the lack of mechanisms for their elimination, which is confirmed by our findings on the decrease in the killing activity of the effector link of phagocytizing neutrophils of peripheral blood.


Fluorescence diagnostics is one of the promising methods for intraoperative detection of brain tumor boundaries and helps in maximizing the extent of resection. This paper presents the results of a pilot study on the first use of the chlorin e6 photosensitizer and a two-channel video system for fluorescence-guided resection of pituitary adenomas. The study’s clinical part involved two patients diagnosed with hormonally inactive pituitary macroadenomas and one patient with a hormonally active one. All neoplasms had different sizes and growth patterns. The data showed accumulation of chlorin e6 in tumor tissues in high concentrations: Patient 1: 2 mg/kg, Patient 2: 5 mg/kg, and Patient 3: 4 mg/kg. For Patient 1, the residual part of the tumor was not resected since it was intimately attached to the anterior genu of the internal carotid artery. For Patients 2 and 3, no regions of increased Ce6 accumulation were detected in the tumor foci after resection. Therefore, the use of the Ce6 and a two-channel video system helped to achieve a high degree of tumor resection in each case. Pituitary adenomas (PAs) are benign neoplasms and account for 10–15% of all intracranial primary brain neoplasms [In recent years, endoscopic transnasal transsphenoidal access has become the gold standard in the surgical treatment of patients with PAs, almost completely replacing transcranial and transnasal approaches performed with a microscope ,3. The eIn recent years, new intraoperative navigation methods, including fluorescence diagnostics (FD), have been applied in neurosurgical practice ,7,8,9. FRegistration of the PS fluorescence allows to more accurately determine the neoplasm boundaries, as well as residual foci of fluorescence as the main part of the tumor is removed ,11.There are several PS that are approved by the FDA for clinical use that have various accumulation mechanisms—cellular or vascular . In seveAt the moment, indocyanine green (ICG) is mainly used as a PS for fluorescence-guided resection of PAs ,17,18. IChlorin e6 (Ce6) and its derivatives are promising PSs with high diagnostic and therapeutic efficiency in neuro-oncology ,21,22. CIn connection with the above, the aim of this work was to evaluate the possibility of clinical application of chlorin-type PS and a two-channel video-fluorescent system for FD of PAs. The main tasks were to determine the degree of the PS accumulation in tumor tissues and to assess the extent of PAs resection using this technology.A two-channel video-fluorescence system  consisting of a white light source, a laser source with a radiation wavelength of 635 nm wavelength, a system of optical filters, and a rigid endoscope with a 45° tilt of the distal edge was used for conducting FD . This syThe metrological characteristics of the system were determined on optical phantoms of pituitary tissue with Ce6 in various concentrations  and a scattering medium  registered with the same calibration as in the analysis of tumor tissue a.®, DEKO Company, Moscow, Russia). Ce6 is a second-generation PS widely used in clinical practice, and is characterized by rapid excretion from the body [As part of the study, a chlorin-type PS was used to perform FD of PAs, the active substance of which is the trisodium salt of chloride e6  included in the study had previously undergone surgery with resection of the tumor at different times and had continued tumor growth. After the initial intervention, according to the histological examination, patients were diagnosed with PA, while Patient 3 had a primary PA. Additionally, at the preoperative stage, the infiltrative nature of tumor growth into the surrounding tissues was assumed. All patients were admitted to the clinic in satisfactory condition, without focal neurological symptoms. Patients 1 and 2 of the concomitant diseases had hypertension, and Patient 3 had secondary hypothyroidism. Patients 1 and 2, both in the preoperative period and after the surgery, took hormone replacement therapy for hypopituitarism: hydrocortisone and desmopressin. Patient 3 was taking L-thyroxine for a concomitant disease. None of the patients, either in the preoperative period or after the surgery, took chemotherapy drugs, since at the moment they are not included in the standard of treatment for patients with pituitary adenomas, given the benign nature of these types of tumors. All patients underwent a study of the hormonal profile and MRI of the brain with contrast enhancement with an assessment of the tumor spread and its interaction with the main nerve structures . The pat®) diluted in 100 mL of saline was intravenously injected to the patients at a 1 mg/kg concentration 3–3.5 h before FD. The tumor was accessed using an optical endoscope and a white light source . After the DM was opened, the localization, the size of residual foci, the concentration of the PS accumulated in the tumor focus, and the zones of infiltration by tumor tissue with an increased fluorescence index relative to tissue considered to be healthy were determined using a two-channel video-fluorescence system. The intact anterior leaflet of the DM, lining the anterior wall of the sella turcica, was taken as healthy tissue. As the tumor was removed, the data were recorded into video files and images for further processing. Tumor resection was stopped when the residual part of the tumor and its capsule infiltrated by the tumor tissue were intimately fused with vital structures, such as the internal carotid artery and oculomotor nerves.Ce6 (FotoditazinAfter removal of the main endosuprasellar node, sensitized tissue with intense Ce6 fluorescence was found in the projection of the right cavernous sinus using the FD method a. After After removal of tumor tissue from the saddle cavity and suprasellar component, FD of the first cavernous sinus zone, where the tumor spread according to the MRI, was performed. A high fluorescence index was registered in this area a. DuringAfter removal of the visible part of the tumor node spreading endosupralaterocellarly, FD of the tumor bed was performed. A region with increased fluorescence was detected a. After All patients in the postoperative period felt satisfactory, and there was a positive dynamic of visual disturbances. All patients were discharged on day 3–5 after surgery without complications. Follow-up of the patients was 1.5 months. Chiasmal syndrome regressed in all 3 patients, and Patient 2 partially preserved oculomotor disorders in the form of diplopia. All patients had no signs of nasal liquorrhea or bleeding.Accurate definitions of the PA boundaries and areas of tumor infiltration of the surrounding tissues are key to a high degree of resection, which can increase the likelihood of successful treatment and preserve normal pituitary functions . The autAt the moment, the most commonly used PS for FD of PA is ICG with fluorescence in the near-infrared region ,17,18In the following studies, after bolus injection of IGG solution in patients with histologically confirmed PA, unaffected tissues showed a stronger fluorescent ICG signal than adenoma, whereas in our study, after intravenous administration of Ce6, the high selectivity of the PS accumulation in adenoma tissues was observed. The greatest contrast in the case of ICG was observed 0.5–3 min after administration, which complicates the process of tumor tissue resection due to the rapid removal of ICG.The authors of , during Therefore, the choice of a PS for complete adenoma resection and the possibility of subsequent PDT of residual foci of the PS accumulation is a difficult task due to the characteristics of normal and affected tissues. It should be taken into account that the permeability of the blood–brain barrier in the pituitary gland is high, so the molecules of Ce6 can quickly diffuse between the vascular and interstitial spaces. There are also significant differences in microvascular density between normal and pathological pituitary tissues .Since Ce6 mainly accumulates in the vascular system of neoplasm tissues, this PS is most often used for PDT. Selective light exposure to the zones of Ce6 accumulation primarily destroys the blood vessels feeding the tumor, since they are more prone to thrombosis. The Ce6 antitumor effect also includes damage to both pathological cells by the mechanism of necrosis and/or apoptosis, and tumor-associated macrophages ,35. The Normalization of the fluorescence index was performed by assigning the fluorescence index value of 10 to the DM lining, which allowed to intraoperatively identify areas with increased fluorescence intensity of Ce6 in all three patients. The highest value of the fluorescence index for Patient 1 was 62 ± 5 rel.u., for Patient 2—130 ± 5 rel.u., and for Patient 3—94 ± 5 rel.u.After the adenoma removal, video-fluorescence navigation showed a significant decrease in the fluorescence index in all areas, which characterizes the absence of residual foci and a high degree of resection. The obtained results are very encouraging and indicate a high selectivity of the Ce6 accumulation in the PA tissues, which gives prospects for further PDT of sensitized residual pathological foci of PA.This paper presented clinical results of the first use of Ce6 PS and a two-channel video system for fluorescence-guided resection of PAs performed on three patients with different tumor sizes and growth patterns. After a comparative analysis of the fluorescence indexes registered on optical phantoms with Ce6 and data recorded during tumor resection in each case, the accumulation of the PS in pathological tissues was determined: Patient 1: 2 mg/kg, Patient 2: 5 mg/kg, and Patient 3: 4 mg/kg.In the first case, the residual part of the tumor with an increased fluorescence index was intimately attached to the anterior genu of the internal carotid artery and was not resected. In the remaining parts of the tumor bed, no increased fluorescence signal was observed. For patients 2 and 3 at the end of resection, no regions with Ce6 accumulation were detected.Since the degree of radicality of tumor resection is of particular importance in hormone-active pituitary adenomas, as well as in recurrent tumors, the routine use of the FD method will increase the number of patients with normalization of hormonal status and remission of the disease. Additionally, a high level of Ce6 accumulation in pituitary adenoma tissues and further fluorescence-guided resection can help to predict the possibility of recurrence of the disease and the need for additional treatment, in particular, PDT in patients with foci of infiltrative growth, accumulating PS, if removal is impossible.


In the comparison TIME A with TIME B, we observe a reduction in the total number of tests performed (46%) and in the number of diagnoses (34%). In both periods analyzed, mucopolysaccharidoses  was the most frequent LD suspected and/or confirmed. Our data indicates a large reduction in the number of tests requested for the investigation of IEM and consequently a large reduction in the number of diagnoses caused by the COVID-19 pandemic leading to a significant underdiagnosis of IEM.The COVID-19 pandemic led to the reorganization of health care in several countries, including Brazil. Inborn Errors of Metabolism (IEM) are a group of rare and difficult to diagnose genetic diseases caused by pathogenic variants in genes that code for enzymes, cofactors, or structural proteins affecting different metabolic pathways. The aim of this study was to evaluate how COVID-19 affected the diagnosis of patients with IEM during the first year of the pandemic in Brazil comparing two distinct periods: from March 1 There aSince 1982, the Laboratory of Inborn Errors of Metabolism of the Medical Genetics Service (LIEM - MGS) of the Hospital de Clínicas de Porto Alegre (HCPA) has contributed to the biochemical understanding of many rare diseases and has become a reference center for the diagnosis of this group of diseases, receiving samples from all of Brazilian states and from other countries. The main specialty of LIEM-MGS is the diagnosis of Lysosomal Disorders (LDs), but the laboratory also frequently performs other tests for IEMs such as the diagnosis of classic galactosemia, type I tyrosinemia, biotinidase deficiency, congenital disorders of glycosylation, Smith-Lemli-Opitz Syndrome, among many other conditions.The main aim of this study was to evaluate how COVID-19 affected the diagnosis of patients with IEM throughout the first year of the pandemic in Brazil.st, 2019 to February 29th, 2020 (TIME A) and from March 1st, 2020 to February 28th, 2021 (TIME B). Data such as the number of tests performed on urine samples (U), whole blood (WB), leukocytes (L), plasma (PL), dried blood spots (DBS), serum (S), fibroblasts (F) and the number of confirmed diagnoses per year performed by LIEM-MGS were also used in this analysis. We considered as a “confirmed diagnosis” those that are performed using the gold standard, usually performed via measurement of the enzymatic activity and/or specific biomarkers in leukocytes and/or fibroblasts, using fluorimetric or colorimetric assays, electrophoresis, chromatography, isoelectric focusing and other types assays/tests, and also cases with a positive molecular genetics diagnosis. Positive results in a single DBS sample were considered a diagnosis “to be confirmed”, and only considered as a “confirmed diagnosis” when analyzed in two different samples or combined with a positive molecular genetics results. The tests and diagnosis were divided into two different groups: Lysosomal Disorders (LDs) and other IEM (IEM-others).This is an observational study in which we compared the periods from March 1Mean and standard deviation were analyzed by SPSS version 22.0.During TIME A the LIEM-MGS performed 7,422 specific tests for IEM. Of these, 6,750 (91%) were requested for IEM-LD investigation, the remaining 672 (9%) for IEM-OTHERS investigation. In TIME B the total number of tests performed for IEM investigation was 3,980. Of these, 3,497 (88%) are represented by IEM-LDs and the remaining 483 (12 %) were tests requested for IEM-others . In the In TIME A, the laboratory was able to diagnose 242 patients for 38 different types of IEM. Of these, 151 (62%) were confirmed and 91 (38%) required further confirmation in an additional sample. Of the 151 confirmed, 142 (94%) were IEM-LDs and 9 (6%) IEM-others. Of the 91 diagnoses that required further confirmation, 87 (96%) represent IEM-LDs and 4 (4%) IEM-others . The meaAmong the diagnoses of IEM-LDs, the mucopolysaccharidoses (MPS) represent 51% (77) of the confirmed diagnoses and 52% (45) of those requiring confirmation. Of the IEM-others, 44% (4) are from patients confirmed with type I tyrosinemia and 75% (3) patients with possible congenital disorders of glycosylation (to be confirmed).In TIME B, 158 patients were diagnosed for 32 different types of IEM. Of these, 99 (63%) were confirmed and 59 (37%) required confirmation in an additional sample. Of the 99 confirmed patients, 91 (92%) are IEM-LDs and 8 (8%) are IEM-others. Of the 59 diagnoses that required confirmation, 56 (95%) represent IEM-LDs and 3 (5%) IEM-others . The meaAmong the diagnoses of IEM-LDs, MPS represented 48% (44) of the confirmed diagnoses and 36% (20) of those requiring confirmation. Of the IEM-others 37% (3) are from patients confirmed with Smith-Lemli-Opitz Syndrome.Comparing both periods we can observe that the number of diagnoses was reduced by 34% (confirmed and/or to be confirmed) with a reduction of 36% in the confirmed cases and 34% of cases needing a confirmation. In the case of IEM-others the reduction in the number of diagnoses was of 11% in confirmed cases and 25% for cases that required confirmation. In both periods analyzed, MPS  represented the most frequent IEM-LDs confirmed and/or to be confirmed. For the confirmed diagnoses for IEM-others the most frequent disorders were type I tyrosinemia in TIME A and Smith-Lemli-Opitz Syndrome in TIME B.The tests requested for the investigation of IEM-LDs had a total reduction of 48%. The most striking reduction was in the tests for the investigation of MPS type III . The assay with a smaller reduction of request was the chitotriosidase enzyme assay (19%) and the assay for the analysis of the lysosomal acid lipase (16%). There was no reduction in the number of requests for the investigation of α-fucosidase and α-N-acetylgalactosaminidase . There was an increase of 50% in the number of requests for the β-mannosidosis assay (from 2 to 4) and of 5% for α-galactosidase A assay .In the tests requested for the investigation of IEM-others, the total reduction was 28%. The greatest reductions were the Erlich test (<50%) and the assay of orotic acid (<44%). The assay for classic galactosemia, by the measurement of the activity of galactose-1-phosphate uridyl transferase had the smallest reduction (<23%) and the requests for the investigation of biotinidase increased by 11% .It is important to emphasize that in Brazil very few laboratories perform the investigation of IEM and that the LIEM is the reference center for these diagnoses. According to a survey carried out by EURORDIS through the Rare Barometer Programme, from April 18, 2020 to May 11, 2020 with 6,945 respondents in 36 countries in Europe with 1,250 different types of rare diseases, 83% of patients had some type of interruption in treatment and 6 out of 10 patients had no access to diagnoses (blood or imaging tests) during the pandemic . In BrazIn a study comparing a 3-months period between 2019 and 2020 in 16 diagnostic and treatment centers for IEM in 11 different countries with approximately 8,500 patients with IEM, it was shown that the laboratories had a reduction of 60% in the number of samples tested and of 80% in the number of new diagnoses. This reduction was even larger that the reduction we have seen in LIEM-MGS  , reportsUnfortunately, the IEM were not the only underdiagnosed diseases in this period. In the Jiangsu Province, China, tuberculosis notifications from January to May 2020 were reduced by 36% when compared to the same period in 2019 . In the Our group had already established the estimated frequency of LDs in Brazil in a study analyzing all diagnosis of IEM made at the LIEM from 1982 to 2015 in which 72% of these diagnoses were represented by IEM-LDs and 27% of these were mucopolysaccharidoses . Our curThe drastic reduction in the number of requests for the investigation of MPS III can be explained by the usual diagnostic challenge, particularly in the early disease stages that can sometimes be confused with autism spectrum disorder, delaying the diagnosis of this disorder regardeless the COVID-19 pandemic . Other fThere was also a decrease in the number of requested tests for the diagnosis of IEM-LDs and IEM-others . This smaller reduction in the number of tests requested for IEM-others as the drop in the average age from 6.3 to 1.2 years in patients with other types of IEM may be explained by the acute and more severe manifestations  and also because of the Federal newborn screening program that screens for diseases such as galactosemia and biotinidase deficiency.When we compare the diagnoses geographically, we do not observe a great variation in the distribution within the Brazilian regions. The reduction in the number of samples sent from other countries, such as Algeria (<69%) and Ecuador (<65%) can be explained probably due to the borders being closed during the pandemic. A questionnaire filled by physicians in the Saab study suggests that in countries such as Algeria, the abandonment of cancer treatment by some patients may be related to limitations in locomotion to treatment centers and the socio-economic impact of the COVID-19 pandemic in these families (Our data indicate that in TIME B (COVID-19 period) there was a dramatic reduction in the number of test requests for the diagnosis of IEM and, consequently, in the number of diagnoses compared to TIME A (the period immediately before COVID-19). As there is no meaningful reason for a drop in the actual number of IEM cases, we conclude that during the COVID-19 pandemics there was a significant underdiagnosis of IEM. Health authorities should act to mitigate these negative impacts of the COVID-19 pandemic as IEM patients need to be promptly diagnosed and regularly monitored in order to benefit from the specific and general management measures available for these conditions.


Mine gas disasters are a major safety concern in underground coal mining. Protective layer mining is widely used in gas disaster control, but there are limited theoretical and experimental results that can provide guidance for site-specific mining circumstances. Taking the Xinji No. 1 mine as an example, gas disaster treatments were conducted in a new panel with overlying goaf located 85 m above the coal mine and adjacent goaf located at 30 m intervals. This study involved a comprehensive investigation, which included four steps: the selection of the first mining face, gas control and prevention, tracking and investigation, and effect analysis and assessment. The safety strategy focused on gas control planning in new mining areas or panels. The distribution and evolution characteristics of the stress, the gas permeability coefficient and the deformation volume within the protected layer were determined by numerical simulation. The coal deformation, gas emission and extraction effect were analyzed by field observation. The deformation and gas permeability of the coal seam were consistent with the stress evolution, for which the maximum compressional and expansional deformation of 6-1 coal were 18‰ and 28‰, respectively. Gas disaster control and prevention treatment of the mining face produced a significant protective effect on the underlying No. 6-1 coal seam. This work is beneficial for the planning of gas control in successive panels. China is the world’s largest energy consumer. China’s primary energy consumption, of which coal accounted for 56.8% in 2020, will reach its peak in 2030 . Coal reWith the advancement of coalfaces, coal seams are expected to undergo a cycle of stress evolution that involves in situ stress (stress abutment), failure and stress reduction (stress recovery). Similarly, the evolution in the permeability of coal during protective layer mining was described experimentally by Yin et al. in four stages . The decThe effect of the protective layer on mining is related to parameters such as layer spacing, dip angle and lithology of adjacent coal seams, and its protective effect needs to be systematically investigated . Guo et Gas content is used worldwide by coal mines to assess outburst risk ,23. ConvProtective layer mining is widely used in gas disaster control, but limited theoretical and experimental results can provide guidance for site-specific mining circumstances. A new panel in a deep mining area entails the facing of more severe challenges in gas disaster prevention. Thus, the evaluation of gas occurrences and the investigation of mining disturbances in protected coal seams, including evolutions of stress, coal body deformation, and permeability, are the basis for safe mining activities, such as mining engineering design in new areas or levels, and reasonable short-term gas control planning. Taking the Xinji No. 1 mine as an example, a set of systematic investigation plans has been put forward to eliminate the outburst danger and ensure the safe mining of the workface. In addition, the spatial variability of gas permeability in coal seams with protective layer mining is presented through simulation analysis in this paper.Xinji No. 1 mine is located in Huainan coalfield, Anhui Province, China, with an annual output of 1.8 million tons. With the geological characteristic of the group occurrence of high-gas coal seams, No. 13-1 and 6-1 coal is in danger of outburst disasters due to the high gas content, and other No. 9, 11-2, 8 coal seams, etc., all have gas occurrences. No. 8 coal seam is extracted in a new mining area (3608), where the coal seam dip angle is 3~15° and its thickness ranges from 2.0 to 4.6 m. The depth of the No. 8 coal seam in the mining area is from 695 m to 810 m, and the No. 6-1 coal seam is from 735 m to 850 m. The layout and histogram of the workface in the mining area are shown in A flow chart for the investigation of the protective layer is shown in The gas occurrence conditions of the coal seam group were evaluated initially, and priority coal seam was selected as the protective layer. The gas parameters for each coal seam are shown in No. 8 coal, with a small gas hazard level, was selected as the upper protective layer. The main reasons were as follows: firstly, the upper protective layer was preferred for mining, as it produces a pressure-relief effect and does not damage the coal seam as a result of rock strata movement; secondly, compared with No. 6-1 coal, No. 8 coal has less potential risk of gas disaster, and has better economic benefits from the perspective of resource reserves. The location of the first mining face is usually selected where geological conditions are representative or gas disaster is easy to be controlled.The 360804 workface, with the overlying goaf located above and the adjacent goaf located nearby the coalmining area, was selected as the first mining face. The overlying goaf formed as a result of the No. 11-2 coal mine in 2011, and is located above the No. 8 coal mining area. The adjacent goaf was formed as a result of the No. 8 coal mine to the south of the mining area in June 2007, and is blocked by a coal pillar of 30 m width. The adjacent and overlying goaf produce a pressure-relief effect that can be conducive for gas control in the 360804 workface.q is the relative gas emission rate in the coalface, m3/t; q1 and q2 are the gas emission rates in the mining seam and the adjacent seam. K1, K2 and K3 are the rock emission coefficient, coal drop coefficient and preparatory workings discharge coefficient, respectively. m and M are the seam thickness and mining height. w0, wi0, cw and ciw are the original gas content of the mining coal seam, the original gas content of the ith adjacent seam, the residual gas content of the mining coal seam, and the residual gas content of the ith adjacent seam, respectively, m3/t. iη is the emission ratio of the ith adjacent seam, %. All parameters were selected as shown in q1 is 1.8 m3/t, q2 is 2.6 m3/t and q is 4.4 m3/t.A predictive method based on different gas sources was used to calculate the gas emissions in the process of coal mining, including the gas emissions from the mining seam and the adjacent seam (refer to the Safety standard of China ). The caGas control in the mining face mainly included reducing the gas flooding into the workface and eliminating outburst disasters in the protected layer. For the first mining face, a conservative method was adopted for gas disaster control. The post-drainage technology ,31 was a3/t by coal bed degassing. It could preliminarily be obtained by empirical method and numerical simulation method, and then determined by field verification method. The empirical method mainly refers to Appendix [As calculated above, the gas emission from adjacent coal seams accounted for a large proportion of the overall emissions. Thus, the outburst disasters of the 6-1 coal seam needed to be considered and controlled. Determination of the pressure-relief range for 6-1 coal, described by the pressure-relief angle, was an important link, as shown in Appendix  of the RThe most fundamental method of gas disaster control is gas extraction. Utilizing the mining effect of the No. 8 coal seam, the pre-drainage technique assisted in the coal mining process by reducing the gas escaping to the workface above and eliminating the outburst risk for the No. 6-1 coal seam. The crossing holes were prepared for drainage of methane and fully covered the vertical projection range of the 360804 workface. The interval between the hole bottoms was 20 m in the transverse and longitudinal directions, as shown in The research on the mining effect mainly includes the mine’s ground pressure, the gas emission of the coalface, the deformation of the protected coal body, the permeability coefficient of the protected layer, the extraction effect, etc.θ is the installation drilling angle, °; α is the dip angle of the coal seam, °; l1 and l2 are the displacements of the deep and shallow base points, m; H is the thickness of the coal seam, m.Observation of the deformation of the coal body was carried out in the floor roadway. The crossing hole of No. 6-1 coal was constructed to install the multi-point displacement meter, as shown in 2/(MPa2·d). A crossing hole was drilled into the coal seam from the roadway. The borehole was sealed and equipped with a pressure gauge until the values of the gas pressure were stable. After the pressure gauge was removed, the gas flow was observed with a flowmeter within a few days. The stable rate of flow was used to calculate the permeability coefficient [2/MPa2·d. The relationship between the permeability coefficient and coal permeability is expressed as follows:2/(MPa2·d); k is the permeability of the coal seam, m2; μ is the dynamic viscosity, Pa·s; np is the atmospheric pressure, Pa. Due to pressure relief and degassing, the λ of No. 6-1 coal in the condition of coal mining was difficult to measure but it could be investigated by numerical simulation . For the mining of the 360804 workface, a compaction area was formed in the middle of the goaf, where the stress was restored and the gas permeability was gradually reduced. 2/(MPa2·d), which was 600 times that of the original state.Curves representing the stress and gas permeability coefficient within the No. 6-1 coal seam were used to describe mechanical response during mining . The ovePoint D and E were chosen to present the evolution of mining disturbances for 6-1 coal c. With tThe results of the field observation of the No. 6-1 coal deformation are shown in The variation of the gas emission in the workface is shown in In the process of mining, there was a significant relationship between the strata behavior and the gas emission. 3/min and 9%, respectively. With the post-drainage technology adopted on the workface, the gas flow rate and gas concentration caused by high-level drilling and directional drilling gradually decreased from 3.8 to 2 m3/min and from 11% to 3%, respectively. It can be concluded that pre-drainage of No. 6-1 coal is the key to preventing gas emission in the workface and controlling outburst disasters.Variation curves for the gas extraction quantity and concentration are shown in 3/t, and the gas pressure was 0.1 MPa. The treatments for the outburst disaster of the No. 6-1 coal seam were very effective.After the stopping of the 360804 coalface (about one year), the gas content and pressure within the No. 6-1 coal were tested and the measurement points were located at A and C in By conducting a comprehensive investigation process for the 360804 workface, the regularity of mining disturbances was well described, especially the variation of the gas emission and the evolution of the permeability coefficient. With the overlying goaf and the adjacent goaf, the coal mining had a remarkable pressure-relief effect on the underlying No. 6-1 coal. The important parameter values, namely the pressure-relief angles in dip and strike direction, were obtained to describe the pressure-relief range. The risk of outburst disaster in the No. 6-1 coal seam and gas emission in the mining face were controlled effectively. According to the evolution of the 6-1 coal deformation and the permeability coefficient, the control efficiency of gas disasters was improved with the increasing of the scope of mining disturbance. The occurrence of gas within the scope of the mining disturbance was conducive to outgassing.l1 and l2 in The gas pressure and content of the 6-1 coal seam in the mining area were statistically analyzed to predict the gas occurrence trend. The safety-line method, for the prediction of gas pressure, has been applied in numerous coal mines in China . As show(1)2/(MPa2·d).The regularity of permeability within the protected layer was verified spatially by means of coal deformation and stress evolution. The maximum compressional and expansional deformation of the 6-1 coal were 18‰ and 28‰, respectively. The gas-permeability coefficient of the 6-1 coal underwent a decrease, an increase and finally stabilized. The high-permeability-coefficient area presented an “O-shape” distribution in the coal seam, and the maximum value was 55.8 m(2)Applying gas disaster control and prevention treatments to the 360804 mining face produced significant protective effects on the underlying No. 6-1 coal seam. The residual gas content and pressure in the area, determined by the dip-pressure-relief angle of 78° and the strike-pressure-relief angle of 58°, were reduced to far less than the threshold values. The treatments, utilizing the mining effect and adopting the pre-drainage technology of crossing holes, played a very effective role in reducing the gas emission in the mining face and eliminating the outburst risk of 6-1 coal.(3)The procedures, methods and results of the investigation are instructive for subsequent applications in other work faces with similar geological conditions. Reasonable prediction of gas occurrence can further improve the guiding role of the investigation results. All of this adds up to a safety strategy that is beneficial to the planning of gas control in successive panels.Aiming at the control of gas disasters in new deep-mining areas, this investigation adopted the methods of theoretical analysis, numerical simulation and field testing to study the control model for safety production. The main conclusions drawn from the investigations are summarized below:


The budding and division of artificial cells engineered from vesicles and droplets have gained much attention in the past few decades due to an increased interest in designing stimuli-responsive synthetic systems. Proper control of the division process is one of the main challenges in the field of synthetic biology and, especially in the context of the origin of life studies, it would be helpful to look for the simplest chemical and physical processes likely at play in prebiotic conditions. Here we show that pH-sensitive giant unilamellar vesicles composed of mixed phospholipid/fatty acid membranes undergo a budding process, internally fuelled by the urea–urease enzymatic reaction, only for a given range of the membrane composition. A gentle interplay between the effects of the membrane composition on the elasticity and the preferred area difference of the bilayer is responsible for the existence of a narrow range of membrane composition yielding a high probability for budding of the vesicles. One of the most ambitious goals of the recently born discipline systems chemistry is the creation of synthetic life following a bottom-up approach ,2,3,4. Ssn-glycero-3-phosphocholine (POPC)/oleic acid (HOA) GUVs can be utilized to drive and govern the division process [s = [POPC] + [HOA]) and the relative ratio between the two components (The most common model for the investigation of the division and budding processes are giant unilamellar vesicles (GUVs) stabilized by an amphiphilic bilayer made of phospholipids and/or fatty acids. In general, division occurs when the volume of the vesicles decreases (mostly due to an osmotic shock) and concurrently the inner surface area of the inner bilayer leaflet decreases in respect to the outer one. Recently, we reported that a pH change occurring inside the lumen of mixed 1-palmitoyl-2-oleoyl- process ,31. In p process ,33,34 in process ,41,42,43sn-glycero-3-phosphocholine, POPC  and oleic acid, HOA (Sigma Aldrich) in mineral oil (M5904 Sigma Aldrich) is allowed to incubate to make a monolayer. The inner solution is prepared pipetting by hand a water in oil microemulsion. The aqueous phase of the inner solution is composed of the enzyme urease , sucrose (Sigma Aldrich), the fluorescence probe pyranine (Sigma Aldrich) and water, while the apolar phase contains POPC and HOA in mineral oil (we used the same concentration of interface). After the period of incubation, the water in oil microemulsion is poured over the interface and centrifuged. The formation of the vesicles takes place at the interface of the two phases, where emulsion droplets sink by gravity in the outer phase and are coated by the amphiphilic molecules to form bilayers (sucrose and glucose are used to enhance the difference of density between the droplets and the outer solution). Details are reported in For GUVs preparation and encapsulation of chemical species, the phase transfer method ,44,45,46The lipid content in vesicles samples was determined by an HPLC apparatus (Agilent 1200 Infinity) by using a C18 column  . HPLC exW). In this model not just the local principal curvatures  ) represents the set of reaction rates involving X, and [X]To describe the pH change inside the GUVs and obtain h is the distance between the two neutral surfaces. The number of molecules of POPC is considered constant. The logistic function in the last term of Equation (9) accounts for the pH-dependent formation of oleate aggregates in the vesicles lumen when a critical pH is reached (pHThe change of the number of molecules of oleate in the inner and outer leaflets is linked to the pH dynamics described by  through The preferred area difference ((t)/(4π) .The model was described in details in ref.  and a sh0 + [HOA]0 (s) and proportions [HOA]0/[POPC]0 (2)2]0 = 6.0 In a typical experiment, a fast enzymatic reaction inducing a pH change inside the GUVs with the consequent division of the vesicles, was obtained in the following conditions: urease (0.5 U/mL), pyranine (5.0 es lumen ) due to es lumen , we demos /HOA [sn-glycero-3-phosphocholine (DPPC)/HOA [Therefore, to fully understand the effect of OPC)/HOA  and 1,2-PPC)/HOA  mixturesW: K, which accounts for the elasticity of the membrane. The reduced volume To gain more insight into the dependence of the equilibrium limiting shapes of a vesicle, and hence the budding probability, on the membrane composition, we studied the influence of mulation a,b for eion of α c. As expK on the membrane composition we defined an arbitrary linear function, K to decrease. Thus, K varies in the interval 4.25–0.85 that represents a reliable range for phospholipids-based bilayers [To account for the dependence of bilayers ,40,54.K and K, were calculated and used as input in SE together with the fixed According to the experimental observations, the budding limiting shape is a compromise between the increase of the reduced preferred area difference caused by the pH increase, with the consequent deprotonation of the inner leaflet, and an optimal elasticity of the membrane. We then used the values of W yields the reduced area difference The minimization of the energy functional  be ∼1.4 ,38,40,42The division process is one of the fundamental function necessary for the self-replication and self-reproduction of biological entities. In modern cells replication is driven by a complex mechanism of growth and division, regulated by proteins and involving several chemico-physical dynamics  ,56. Howeter GUVs ,31. Inteter GUVs .In this paper we presented some experiments that showed how the mechanical properties of the membrane are another ingredient necessary to understand the physical basis of protocells division. In fact, by varying the ratio between phospholipids and fatty acids, we could control both the ratio of the membrane’s bending moduli and the number of mobile molecules in the inner leaflet of the bilayer . We thus demonstrated that, for a mixed POPC/HOA vesicle, an effective budded shape could be attained only in a narrow range of the membrane composition, where the elasticity and the preferred area difference of the bilayer assume optimal values. According to experiments and simulations the budding region is comprised in the interval


Senecavirus A (SVA) is a newly porcine virus that has been detected in many countries since its first detection in pigs in Canada in 2007, and it remains endemic in many countries in Asia and America, which has become a substantial problem for the pig industry. Vaccination is a potentially effective strategy for the prevention and control of SVA infection. Our lab has developed a SVA vaccine candidate previously. In this study, the antibody response to the prepared vaccine in sows and their offspring was evaluated. Vaccination of sows with inactivated SVA vaccines during pregnancy elicited SVA-specific virus-neutralizing antibodies. Vaccination with a high dose of SVA vaccine followed a booster immunization contributed to a long-term duration of the persistence of maternally derived neutralizing antibodies (MDAs) in the milk of the sows (>14 days). In contrast, vaccination with a single low dose of SVA vaccine resulted in a short-term persistence of MDAs in the milk (2–7 days). The MDAs could be efficiently transferred from the sows to their offspring through the colostrum/milk but not the umbilical cord blood. The antibody titers and the duration of the persistence of MDAs in the offspring are highly associated with the antibody levels in the milk from the sows. Vaccination of sows with a booster dose of SVA vaccine resulted in a longer-lasting MDAs in their offspring (persisted for at least 90 days). However, vaccination with the single low dose of vaccine only brought about 42 days of MDAs persistence in their offspring. The effect of MDAs on active immunization with SVA vaccine in offspring was further evaluated, which showed that vaccination of the SVA vaccine in the presence of MDAs at the titer of ≈1:64 or less could overcome the MDAs’ interference and give rise to effective antibody response. This will help for establishing the optimal times and schedules for SVA vaccination in pigs. Senecavirus, family Picornaviridae. As the only member of genus of Senecavirus, although SVA contains a typical picornavirus L-4-3-4 genome layout, its viral genes differ remarkably from those of all other picornaviruses [pro, 2A, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol [Senecavirus A (SVA), also known as Seneca valley virus, belongs the genus of aviruses ,2. SVA gnd 3Dpol .SVA infection causes typical porcine idiopathic vesicular disease manifested by ruptured vesicles and erosions in the oral cavity, vesicle lesions on snouts and coronary bands, as well as lameness , which aSVA infection in pigs only sporadically occurred in the US and Canada before 2014 ,12. HoweAll the animal experiments described in this study were approved and implemented according to the requirements and management guidelines of the Gansu Animal Experiments Inspectorate and the Gansu Ethical Review Committee (License no. SYXK (GAN) 2017-003).The schematic diagram of the animal experimental design is shown in BHK-21 cells were used to multiply SVA (CH-FJ-2017 strain). The clarified SVA cultures were inactivated using the aziridine compound binary ethylenimine (BEI) to prepare viral antigens as described previously . The inaTen days before mating, 9 healthy sows (SVA-seronegative) with similar age and body weight were divided randomly into three groups: (1) the PBS control group , (2) the LMA group , and (3) the HMA group . As for the immunization method, all the pregnant sows were vaccinated intra-muscularly in the neck with different doses of SVA vaccines as described in the “Study design” section. Blood was collected via jugular venipuncture from the sows on the day 15 before farrowing and on the day of birth, respectively. The umbilical cord blood was also obtained on the day of birth. The neutralizing antibodies in the blood were detected by the VNT. The colostrum and the milk from the sows were collected on day 0, 1, 2, 3, 6, 7, and 14 after farrowing. The neutralizing antibodies in the colostrum/milk were determined by VNT. The MDAs levels in all of the piglets right after intake of colostrum was measured and monitored before getting vaccinated. The offspring of the sow NC #1 (in the PBS control group) and no. #4 (in the HMA group) were immunized with SVA vaccines (2 μg of antigen) at 30 days of age. The offspring of the sow NC #2 and no. #5 were immunized with SVA vaccines (2 μg of antigen) at 90 days of age. The offspring of sow NC #3 and no. #6 were immunized with SVA vaccines (2 μg of antigen) at 120 days of age. The antibody responses in the vaccinated piglets were evaluated by VNT after vaccination.50) was added to all wells except for the control wells. Then, the mixtures were incubated at 37 °C for 1 h in a CO2 incubator. Then, 50 μL of diluted BHK-21 cells (106 cells/mL) in MEM medium supplemented with 8% FBS were added to each well. Endpoint titers were calculated at 72 h post-infection (hpi) and expressed as the reciprocal of the final dilution that led to the neutralization of the virus activity by 50%.The SVA-specific neutralizing antibody titers in the milk and serum of the sows and their offspring were measured by virus neutralizing antibody test (VNT). The VNT was carried out using the method described previously ,28,29. B9TCID50/mL) by intranasal routes as previously described [The vaccinated pigs were infected with 3 mL of SVA  following the protocol provided by the manufacturer. The cDNAs were synthesized using 2 μg of the extracted RNAs as templates. The reverse transcription reactions were performed using random hex-amer primers and M-MLV reverse transcriptase . Then, the cDNAs were detected using the quantitative PCR method in a Mx3005P qPCR System (Applied Biosystems) as described previously . The CT The serum samples collected from all the sows of three groups  at 15 days before farrowing were subjected to VNT analysis. The vaccination-triggered antibody responses were observed in all of the sows in both the LMA group and HMA group. The sows in PBS control group showed no SVA-specific neutralizing antibody responses A. The soTo monitor the dynamics of MDAs in milk  of the sows throughout lactation, the colostrum (day 0) and milk on day 1, 2, 3, 6, 7, and 14 after farrowing were collected respectively, and the neutralizing antibody titers were evaluated by VNT. No specific SVA antibodies were detected in the colostrum and milk from the sows in the PBS control group A. In theNeutralizing antibody levels in milk from sows were strongly associated with the antibody titers in their offspring. Without MDAs, the piglet will be highly susceptible to infection. The piglets were weaned at 28 days of age. The antibody titers in the offspring (serum samples) of all the sows used in the experiments were assessed. No SVA-specific antibody was detected in the serum of piglets from the PBS control group A. The SVTo evaluate the interference of the MDAs to the adaptive immune responses in the piglets, three litters of pigs in the HMA group were immunized with SVA vaccines at 30, 90, or 120 days of age. The offspring from sow no. #4 was vaccinated at 30 days of age. The offspring from sow no. #5 was vaccinated at 60 days of age, and the offspring from sow no. #6 was vaccinated at 120 days of age. The neutralizing antibody titers were measured at different time-points post vaccination. The three litters of pigs in the PBS control group (SVA-seronegative) were subjected to the same treatment and used as the controls for the HMA group. Comparing to the piglets from the control group, the MDAs apparently caused interference to piglet humoral response development after immunization with SVA vaccine at 30 days of age A. The coThe viral infection assay was also carried out in the vaccinated offspring . The offThe outbreaks of SVA infection in pigs have been increasing in many countries and regions in recent years, which has become a substantial problem for the pig industry . SVA infMaternal antibody interference is independent of the type of vaccine being used. A cross-link between B cell receptor (BCR) with the Fcγ-receptor IIB by a vaccine–antibody complex results in the B cell inhibition ,36. TherWe also confirmed that the SVA antigen immunization-induced specific neutralizing antibodies were only transferred from sows to the offspring through drinking colostrum and milk but not through the umbilical cord blood on the day of farrowing . TherefoAlthough the MDAs are crucial for offspring to prevent early viral infections, when the antibody titer declines to a very low level, it will not provide complete protection against viral infections. Hence, the adaptive immunization is essential for strengthening antibody response in offspring. However, the interference by high titers of MDAs prevents the development of adaptive immune response . The preIn conclusion, our study has determined the kinetics of MDAs in the colostrum/milk from sows throughout lactation and the persistence trajectory of transferred antibodies in the offspring. Meanwhile, we evaluated the interference of MDAs on adaptive immune response in the offspring and identified the optimal time point for getting vaccination to overcome the interference. This will help establish appropriate immunization schedules for using SVA vaccine in pigs.


The Mo coating had better microhardness, wear, and corrosion resistance than substrate, which could significantly improve the service life of the H13 steel.H13 steel is often damaged by wear, erosion, and thermal fatigue. It is one of the essential methods to improve the service life of H13 steel by preparing a coating on it. Due to the advantages of high melting point, good wear, and corrosion resistance of Mo, Mo coating was fabricated on H13 steel by electro spark deposition (ESD) process in this study. The influences of the depositing parameters  on the roughness of the coating, thickness, and properties were investigated in detail. The optimized depositing parameters were obtained by comparing roughness, thickness, and crack performance of the coating. The results show that the cross-section of the coating mainly consisted of strengthening zone and transition zone. Metallurgical bonding was formed between the coating and substrate. The Mo coating mainly consisted of Fe H13 mold steel is mainly used for casting, extrusion, hot forming, and plastic molding applications due to its excellent hot-cold manufacturing properties, dimensional stability, and impact toughness resistance ,3,4. H13Mo is important to the industry due to its high-melting-point, high thermal conductivity, and low thermal expansion coefficient . MoreoveIn this study, the H13 steel was chosen as the substrate with sizes of 10 mm × 10 mm × 5 mm. The chemical compositions of H13 steel are shown in The DZS-1400 ESD system  was used to prepare the Mo coating on the substrate. The microstructure and wear scar morphology and the chemical compositions were observed and analyzed by scanning electron microscope  equipped with an energy dispersive spectrometer (EDS). The roughness and three-dimensional morphology of the coating were tested by the OLS3000 (China) laser confocal microscope. Roughness was evaluated quantitatively by arithmetical mean deviation of profile Ra. The phase constitutions were determined via the X-ray diffractometer (XRD) machine  using Cu Kα radiation.The microhardness of the coating was tested by the HVS-1000 microhardness machine  with a load of 100 g and dwell time of 10 s. The wear resistance of the coating was investigated for the low-speed rotation by the ML-100 abrasive wear test machine . The electrochemical corrosion performance and corrosion mechanism were studied by the VersaSTAAT3 electrochemical corrosion workstation  with a three-electrode system. The saturated calomel electrode was used as the reference electrode and the platinum black electrode as the auxiliary electrode. The potential scanning range was −1.5 V~1 V with the scanning speed of 45 mV/min, and the corrosion medium was 3.5 wt.% NaCl solution. Because the sample was a metal material with electrical conductivity, the non-corrosive area had to be coated with a layer of insulating material to prevent other areas from conducting and affecting the test results.The Mo coating appearance is shown in E is the pulse electric spark discharge energy per unit time, P is the deposition power; f is the pulse frequency, and ont is the single pulse discharge time. According to Equation (1), the greater the deposition power P is, the greater the pulse spark discharge energy E per unit time is. When the deposition power is less than 800 W, the melting amount of the electrode increases with the deposition power. It is difficult to uniformly connect and stack of the deposition points during the deposition process due to the rapid cooling rate of the liquid metal, resulting in the increase of roughness. The melting amount of the electrode increases significantly when the deposition power exceeds 800 W. The flow of liquid metal could fill the concave of the deposited layer, so the roughness of the coating can be reduced.2. 2.The substrate of the 10 mm ×10 mm area is carried out by ESD to prepared Mo coating with different specific deposition time. The specific deposition time is 1~5 min/cm2. The thickness of the coating is uneven when the specific deposition time exceeds 3 min/cm2. At the same time, there are microcracks that appear in the coating. There are many reasons for the occurrence of microcracks. On the one hand, the ESD process is a process of instantaneous heating and rapid cooling. If the liquid electrode material that has transitioned to the substrate surface has not completely solidified, the next pulse discharge will arrive. Thereby, thermal stress is generated in the material. On the other hand, the ESD process is accompanied by thermal diffusion. The heat from the coating surface was diffused along the depth direction of the substrate. Appearance of microcracks in the coating is due to the large temperature gradient during the ESD process.The thickness seriously affects the properties of the coating. The specific deposition time is the crucial parameter that affects the thickness of the coating during ESD. The cross-section images of the coating obtained with different specific deposition time are shown in The frontal images of coating fabricated by different processing parameters are shown in 2. However, the thickness of the coating decreases when the specific deposition time increases to 5 min/cm2. Due to the long specific deposition time, the coating undergoes many times rapid melting and cooling processes. Therefore, the surface of the coating produces thermal stress. It is shown in The effect of deposition power on the thickness of the coating is shown in 2, respectively. Finally, the Mo coating with 35 μm thickness and about 5.0 μm roughness was prepared without severe cracks.The thickness and roughness of the coating are important indexes to evaluate the quality of the coating. The effects of the deposition power, discharge frequency, and specific deposition time on the coating quality were studied, and the optimal parameters were obtained as follows: the deposition power, discharge frequency, and specific deposition time were 1000 W, 350 Hz, and 3 min/cm2) by the ESD process is shown in 9.7Mo0.3, Fe-Cr, FeMo, and Fe2Mo cemented carbide phases. Jia Delong et al. obtained similar XRD analysis results at the plasmas sprayed Mo coating on stainless steel substrate [9.7Mo0.3, FeMo, and Fe2Mo phases were formed by the metallurgical reaction between the Mo electrode material and the H13 steel substrate during the ESD process. It is beneficial for good mechanical properties that the coating was formed by the metallurgical bond between the Mo coating and the H13 steel substrate [The XRD pattern of the coating fabricated under 1000 W/350 Hz/2, respectively.The Mo coating without defects was successively prepared on the H13 steel substrate by ESD process. The effect of main parameters on the thickness and roughness of the coating was studied. The well main parameters were obtained as follows: the deposition power, discharge frequency, and specific deposition time were 1000 W, 350 Hz, and 3 min/cm(2)9.7Mo0.3, Fe-Cr, and FeMo, Fe2Mo cemented carbide phases and amorphous phase. The coating cross-section mainly consisted of the strengthening zone and transition zone. The metallurgical bonding was formed between the coating and substrate.The Mo coating was mainly composed of Fe(3)The microhardness of the coating distribution showed a noticeable gradient, and the microhardness gradually decreased from the coating top surface to the substrate. The wear resistance of the coating was about seven times higher than that of the substrate. The amorphous phase and cemented carbide phase improved the microhardness and strengthened the bearing capacity of the coating.(4)The self-corrosion of the coating potential increased by 9%, and the self-corrosion current density was 24% of the substrate. The corrosion resistance of the coating was much better than that of the substrate due to Mo’s excellent corrosion resistance by ESD technology preparation.