Schizosaccharomyces pombe (fission yeast) have revealed unexpected functions of cytoplasmic microtubules in nuclear architecture and chromosome behavior, and have pointed to NE-chromatin tethers as protective elements during nuclear mechanics. Here, we review and discuss how fission yeast cells can be used to understand principles underlying the dynamic interplay between genome organization and function and the effect of forces applied to the nucleus by the microtubule cytoskeleton.In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in compacted conformation at sites of cytoskeleton contacts seem to be key for withstanding nuclear mechanical stress. Mechanical perturbations of the nucleus normally occur during nuclear positioning and migration. In addition, cell contraction or expansion occurring for instance during cell migration or upon changes in osmotic conditions also result innuclear mechanical stress. Recent studies in  Eukaryotic cells show a complex nuclear organization that has co-evolved along with increasing genome size and complexity. Nuclear organization is essential to many aspects of genome regulation and stability. Regulation of gene expression, DNA replication and repair, and ribosome synthesis depend on positioning and complex interaction of DNA and proteins within the nucleus ,2,3,4. TAn important element in the 3D organization of the nucleus is the nuclear envelope (NE). The NE, inner nuclear membrane (INM) proteins, and other NE-associated complexes such as linker of nucleoskeleton and cytoskeleton (LINC) complexes, nuclear pore complexes (NPCs), or nuclear lamina in animal cells provide a binding platform for specific DNA sequences and chromatin, creating specialized chromatin domains and contributing to the 3D organization of the genome ,14,15,16Schizosaccharomyces pombe has been established as an excellent model organism for the study of conserved principles underlying eukaryotic nuclear organization  with just three chromosomes and a relatively large nucleus, which is suitable for microscopic observation. Its chromatin organization and regulation share many features with that of higher eukaryotes [The fission yeast iewed in ,7,8,9). karyotes ,23,24, akaryotes ,26,27. The fission yeast cytoplasmic microtubule MT cytoskeleton is relatively complex  and, amoIn this review, we will first provide a brief overview of global nuclear organization in the fission yeast with a focus on the role of the NE and its associated elements as genome organizers. Then, we will highlight the latest findings on the role of cytoplasmic MTs on chromosome dynamics, the modulation of the DNA damage response, homologous recombination (HR), and chromatid cohesion. Finally, we will discuss how this overall nuclear organization is relevant to bear mechanical forces produced by cytoplasmic MTs.S. pombe, the three chromosomes are organized in a Rabl-like configuration in which centromeres are clustered and attached to the NE beneath the SPB, and telomeres are attached to distant sites in the NE opposite to the SPB [S. pombe genome. Man1 has a broad interaction map that spans about a third of the genome and its interacting domains are enriched for Swi6/HP1, a hallmark of heterochromatin [In  the SPB  . Cenome arms . This reome arms ,47 . ThusS. pombe, centromere attachment to the NE depends on the centromere-bound protein Csi1 and the LINC complex [S. pombe Lem2 localizes at the NE and is also concentrated underneath the SPB in a Csi1-dependent manner, where it has a role in centromere positioning at the nuclear periphery and also in centromeric chromatin silencing [lem2Δcsi1Δ shows severe centromere detachment and defective pericentromeric gene silencing [In  complex   of the mating-type locus and other dispersed TFIIIC sites [S. pombe is still unknown; however, in S. cerevisiae, ETCs peripheral localization depends on the LINC complex component Mps3 [Transcription boundaries or boundary elements (BEs) are genomic positions that function by isolating heterochromatin domains from the surrounding euchromatin (reviewed in ). BEs art the NE ,67, and t the NE ,66,67,68t the NE ,58 throuIC sites . These eIC sites . To dateent Mps3 . S. pombe as model system. INM proteins tether chromatin at multiple sites immobilizing these regions and also have a role in the regulation of heterochromatin. In the last few years, it has been demonstrated that the state of chromatin impacts the mechanical response of the nucleus. Condensed chromatin provides rigidity to the nucleus and metazoan cells can regulate the level of chromatin compaction in order to increase the resistance of the nucleus in conditions of mechanical stress [The functional significance of the interaction between the INM proteins and chromatin is an on-going field of study where many advances have been achieved using l stress ,73,74,75S. pombe, cytoplasmic MT bundles apply forces to the nucleus during unperturbed conditions [Cells are constantly subjected to mechanical stress in nature, which has profound repercussions not only in cell shape and morphology, but also in nuclear architecture. Nuclear architecture is in turn essential to resist nuclear mechanical stress. Tension, shear stress, or changes in pressure generate mechanical forces that are transmitted to the nucleus and this affects nuclear positioning, shape, and function. In nditions  and theynditions ,36. ImpoS. pombe cells, NE fluctuations in response to MT forces are more prominent in the area close to the SPB, which suggests that this area of the NE is subjected to stronger MT forces. Accordingly, impairment of these SPB-enriched chromatin tethers in the double mutant lem2Δ ima1Δ results in the most pronounced and lasting NE deformations in vivo. This suggests that the regulation of chromatin tethers at the NE might constitute an active mechanism to modulate chromatin flow and the mechanical response of the nuclei to cytoplasmic MT-driven forces [lem2 deletion also affects centromeric chromatin silencing [lem2Δ mutant shows altered nuclear membrane lipid composition [In the last few years, chromatin has emerged as an important element that contributes to the mechanical resistance of the nucleus ,76. Forcn forces . Of noteilencing  and therilencing . The lemposition ,79, thatposition ,81. Thisposition ,83,84,85Importantly, interphase MT depolymerization is thought to be regulated by force- and length-dependent mechanisms ,86,87,88S. pombe, the use of in vivo genomic tagging systems such as the lacO/LacI-GFP or tetO/TetR–tdTomato systems have allowed the study of the behavior of specific chromatin positions in relation to MT movements. These studies have shown that cytoplasmic MTs affect chromosome behavior both during the meiotic and the mitotic cell cycles [In l cycles ,33,34,35S. pombe, the effect of MT-driven movements on chromatin dynamics has been best characterized during meiosis. During meiotic prophase, rapid and extensive nuclear movements are driven by dynein- and MT-dependent pulling forces generated at the cell tips during the so-called “horsetail movement” [In ovement” ,91,92,93ovement” ,92,93. Hovement” ,95. Thusovement” . It is kovement” . Indeed,ovement” . After tovement” .Therefore, meiotic MT-driven nuclear movements promote dynamic chromosome pairing and unpairing to modulate the extent of chromatin contacts and recombination. The lack of MT-dependent movements of chromosomes during meiotic prophase affects spore viability and the efficiency of gamete production . AnalogoS. cerevisiae and Drosophila cells, this relocalization requires proteins of the SUN family [The NE constitutes a protective environment where persistent double-strand breaks (DSBs) are recruited and repaired . In S. cN family ,100 and N family ,102.S. pombe, the LINC complex also contributes to the repair of persistent DSBs [kms1, or mto1, a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus [In ent DSBs . The ind nucleus ,106,107, nucleus . This su nucleus . Budding nucleus ,109.mto1 abolishes chromosome movements and results in altered cycles of chromatin breathing in which chromatids appear more frequently unpaired compared to unperturbed cells [psc3-1T cohesin mutant [mto1Δ mutant cells, in which MT-driven movements of chromatin are abolished, show a significant decrease in the efficiency of intrachromosomal HR. Consistently, mto1Δ cells present increased sensitivity to DNA-damaging agents and defects in HR-based DNA repair [mto1Δ compared to wild-type cells, whereas the levels of Rad21 bound to several other loci at different positions of chromosome arms are significantly reduced [In the fission yeast, the magnitude of nuclear movements during the mitotic cycle is much smaller than during the meiotic horsetail period. Nonetheless, during interphase, cytoplasmic MT bundles also move chromosomes in an oscillatory manner via linkages through the NE at the SPB and other distant sites  Figure A–C. Duried cells . This phed cells ,111,112 n mutant . mto1Δ mA repair . Of note reduced . Mto1 re reduced . This suDuring the past years, the fields of nuclear organization and mechanics have witnessed remarkable progress and have contributed to a better understanding of how genomes are organized in the interphase nucleus, how this is critical for genome functions, and how forces applied to the nucleus can alter this organization and regulate cellular functions. Mechanical forces are intrinsic to many cellular processes. For example, fluid forces and shear stress are known regulators of cardiac development ,114 or i


Pinnularia aljustrelica, Pinnularia acidophila, Pinnularia acoricola and Eunotia exigua, which are the two groups found in the most AMD contaminated dams. Twenty-three water dams located in the Iberian Pyrite Belt were studied during March 2012 (early spring) in order to carry out an environmental assessment based on diatom communities and to define the relationships between these biological communities and the physico-chemical characteristics of the dam surface water. This is the first time that a diatom inventory has been done for dams affected by acid mine drainage (AMD) in the Spanish part of the Iberian Pyrite Belt (IPB). It was found that the pH was the main factor influencing the behaviour of the diatom communities. Then, using a dbRDA approach it was possible to organize the aggrupation of diatoms into four groups in response to the physico-chemical conditions of the ecosystem, especially pH: (1) Maris, Aac, Gos, Cmora (pH 2–3); (2) Andc, San, And, Dpin (pH 3–4.5); (3) Gran, Pleon, Oliv, Lagu, Chan, SilI, SilII, Joya, Gar, Agrio, Camp, Corum (pH 4.5–6); (4) Herr, Diq I, Diq II (pH 6–7). The obtained results confirmed the response of benthic diatom communities to changes in the physico-chemical characteristics of surface water, and helped to understand the role of diatoms as indicators of the degree of AMD contamination in those 23 dams. Special attention was given to those that have an acidophilic or acid-tolerant profile (pH 2–3 and pH 3–4.5) such as  The main problem with sulfide mining is the contamination of water resources caused by acid mine drainage (AMD) processes. AMD occurs when sulfurous minerals are exposed to atmospheric , hydroloIn areas with a semi-arid climate, dam construction is one of the most common solutions to meet the industrial and agricultural water needs of the population. However, the susceptibility of surface water to contamination is much higher than that of groundwater . The proThe mining of metal sulfide ores is one of the main causes of AMD generation due to the oxidation of the sulphide ore, which creates sulphates and hydrogen ions. This is the case in the Iberian Pyritic Belt (IPB) where sulfide ores have been exploited since Roman times, although there is evidences demonstrating that extractive activities could have begun in 4500 years B.P. . The SpaThus, from an ecological point of view, AMD defines extreme environments  e.g., ,11,12) w,12 w11,1Diatoms are one of the main groups of organisms in AMD-affected streams . The WatPinnularia species are highlighted as they are among the few organisms able to live in such extreme conditions.In order to carry out an environmental classification based on the diatom communities, especially those that are acidobiontic or acidophilous, 23 AMD affected dams were selected, in the Spanish part of the Iberian Pyrite Belt (IPB). The dams were divided based on the different purposes for storing water: mining, for industrial use and for human supply. In general, this study intended to assess and evaluate the ecological and physico-chemical status of the water dams affected by AMD, and which store water for different purposes. The specific objectives of this work were (1) to describe benthic diatom communities, (2) to assess their relationship with water physico-chemistry specially the pH, and (3) to understand the effectiveness of diatoms as indicators of the degree of AMD contamination, also, several acidic 2), sphalerite (ZnS), galena (PbS), chalcopyrite (CuFeS2) and arsenopyrite (FeAsS) with other sulfide associated phases of Cd, Sn, Ag, Au, Co, Ni, etc. [The Iberian Pyrite Belt (IPB) has one of the world’s greatest concentrations of sulfide deposits and extends from Lousal  to Aznalcóllar (Spain). The IPB constitutes one of the largest metallogenic regions in the world with approximately 1700 Mt of sulfide reserves . RecentlNi, etc. . Many exNi, etc. . Most of these old mining operations were developed in the absence of environmental regulations for discharges. This has resulted in an extensive river network with extraordinary levels of AMD pollution. In many cases, the pollution remains even hundreds of years after the mine closure, with the Tínto and Odiel rivers being the main receptors of these contaminated leachates. Because of this, these two are the most cited rivers in the scientific literature as paradigmatic AMD contaminated rivers.Most of the 88 inventoried mines from the Spanish part of the IPB  generateThe semi-arid Mediterranean climate of IPB, with an annual precipitation of around 500–700 mm, generates the problem of water insufficiency. In such a climate, and this hydrological and mining context, water dams have emerged as the best solution to solve the problem of water storage and supply. The Spanish Society of Dams and Reservoirs (SEPREM), officially recognizes 30 dams and reservoirs in the Iberian Pyrite Belt, which are publicly and privately owned, and are used for agricultural, and industrial use, or even for urban supply. AMD reaches the dams and reservoirs via the two most affected rivers, Odiel and Tínto  which have a low pH and a high metal load and sulfates. The present work is focused on 23 dams (from the 30 recognized) of the Spanish part of the IPB . They arThe sampling period and the selection of the sampling points  was done+ portable multimeter . After this, two water samples were taken and stored in sterilized polyethylene bottles for each sampling point: one for sulfate determination and the other for metals. Nitric acid was added to obtain a drop to pH < 2 in order to prevent metal precipitation during the transport of the sample to the laboratory in a portable refrigerator, at 4 °C.Determination of the pH, electrical conductivity (EC) and total dissolved solids (TDS) was done in situ using a CrisonMM40In the laboratory, the water samples were vacuum-filtered using 0.45-micron cellulose nitrate filters . Once filtered, the water samples were stored in hermetically sealed polyethylene containers in a refrigerator at 1–4 °C. All the reagents used were of super pure quality . The standard solutions were Merck AA Certificate. Milli-Q water was used in all of the experiments. Sulfate concentration was determined through a Macherey-Nagel PF-11 photometer . Metals and arsenic analysis was done using an atomic absorption spectrophotometer  equipped with a graphite furnace and an air/acetylene-flame atomizer. The samples were introduced using the Perkin-Elmer AS800 Autosampler . Perkin-Elmer Lumina™ hollow cathode lamps (HDL and LDL) were used as sources of radiation.®. Then, diatoms were observed, identified with the highest taxonomic resolution and counted  using two light microscopes: Olympus CH30 and a Leitz Biomed 20 EB  and based on Krammer and Lange-Bertalot [The sampling of diatoms was done according to . The uppBertalot ,39,40,41Bertalot ). OMNIDIPinnularia species (∑ % Pin) were used to summarize all the data on the species at the 23 sampling sites. For the statistical analysis, cluster analysis techniques were applied to the data related to 16 physico-chemical and two biological variables, the sum of number of diatom species (∑ N° Sp.) and the sum of the relative abundance % of These two biological variables were used as a summary in the total matrix of 118 diatom species. Cluster analysis has been widely used for the geochemical characterization of AMD media ,43. ClusThe program used for this analysis (STATGRAPHICS200 Centurion XVI) operates by applying the Ward method or “second-order central moment”, which is a hierarchical method that calculates the mean of all the variables for each cluster; next it calculates the Euclidean distance between each factor and the mean of its group and then adds the distances from each case. In each step, the clusters that are formed are those that yield the smallest increment in the total sum of the intra-cluster distances. In short, through the application of this technique, the variables studied can be classified into different “categories”. Pinnularia species (∑ % Pin) as variables was done, and then a second cluster was done considering the sampling sites as variables. A statistical summary was also obtained with STATGRAPHICS200 Centurion XVI.First, a linear cluster considering the physico-chemical variables and biological data (the sum of number of diatom species (∑ N° Sp) and sum of the % of relative abundance of 42−), the Euclidean distance similarity matrix was calculated. A distance-based redundancy analysis (dbRDA) was performed in order to find linear combinations of the predictor variables, which explain the greatest variation in the data in the total matrix of 118 taxa × 23 samples × 16 environmental variables. Distance-based redundancy analysis (dbRDA) is a method for carrying out constrained ordinations on data using non-Euclidean distance measures. dbRDA circumvents this issue using a three-step process: first, a distance matrix is calculated using the distance measure of choice. Next, a principle coordinates analysis (PCoA) is done on the matrix. Finally, the eigenvalues obtained in the PCoA are plugged into an RDA. SIMPER analysis was used to discriminate the species responsible for the largest contribution to the Bray-Curtis dissimilarity in diatom abundance between the samples within each of the four pH groups first discriminated in the dbRDA.PRIMER v.6   with thePinnularia species (∑ % Pin) at the 23 sites is shown in 42 and metals  in dissolution, which is typical of AMD affected systems.The statistical summary of 16 physico-chemical and two biological variables, the sum of the number of diatom species (∑ N° Sp.) and the sum of the % of relative abundance of Pinnularia species having a higher survival capacity in acidic conditions and greater adaptability to different media, depending on the total species (∑ N° Sp) considered in our data matrix. The % of variance is the parameter that provides the most important information, because it indicates what degree of dispersion each variable can take, respecting the central tendency (average). This fact is of crucial importance, especially for the biological variables ∑ % Pin and ∑ N° Sp.: a high value for the first (161.7%) and low value for the second (48.9%) allowed us to define the conditions that control the survival scenario of the species in this geological region (IPB), with 2+ mobilized from iron sulfide oxidizes to Fe3+. In general, as long as the pH does not reach 3, the ferric ion precipitates as oxyhydroxisulfate, promoting a decrease in its concentration in solution, while the pH drops due to liberating hydrogen ions [3+ concentration increases and acts as a pyrite oxidant without the need for dissolved oxygen, giving the system more Fe2+, SO42− and H+. The most extreme % variance values are the those for Fe and Cu (> 400%). This is shown in gen ions ,47. AccoThe lowest % variance values correspond to Pb, Al, temperature and pH. The reasons for this are very different: Pb only reaches 30%, since it is present in very defined paragenesis of the IPB  and submitted to restricted solubility control conditioned by (pH/Eh) ratios . Al is nAs, Cd, Ni, Co, and Sb did not show significant concentrations in this environment . However, it is important to note that the concentration value of As is high: 1.69 mg/L. It is 169 times higher than the limit allowed by , which iThe low % variance for pH (30%) could be misunderstood when compared with the high % variance shown for As, Co (~300%) as well as for Fe and Cu . The justification for this discrete % variance is the logarithmic character of pH. The dendrograms corresponding to the physico-chemical-biological variables and sampling sites are presented in Al is put on its own on the sub-cluster on the far right, and ∑ Sp., pH and temperature (T.) are together in the other sub-cluster on the right. Al is alone because it is not part of the minerals from the primary paragenesis (pyrite), on the contrary, it comes from the shales of the hosting rocks . This faThe subdivision of the main cluster (on the far left) into two sub-clusters is done according to:∑ % Pin is opposite to high pH and to high ∑ Sp., since ∑ % Pin is closely related to metals  and to acidic water ,11,19). TDS is intimately associated with EC in the sub-cluster that shows the highest Pearson’s proximity ratio. This is common in water without dissolved chlorides, which are responsible for the EC variations. In a higher sub-cluster, EC and sulfates were found in close association with AMD typical metals , as already described by . In thisHigh EC is related to high sulfate content (sulfates dissolution caused by pyrite oxidation), which is typical of AMD contaminated waters. Mn is not in the sulfur paragenesis but in purple schists on the top of the mineralization . Zn is nCd, Ni, Co, Sb do not show important or discriminant behavior, as already mentioned in the previous section. Thus, the divisions in this cluster suggest different contamination causality processes in the hydrological environment.ACluster A: samples from A_Acidas to Marimillas with pH < 5.68;BCluster B: samples from Campanario to Silillos II with pH > 5.70.The dendogram in Cluster A is divided in 3 sub-clusters:(A1) A_Acidas, Gossan, Cueva de la Mora, Del Pino, Grande are close together because of their low pH 2.48–4.10, high metal concentration and high ∑ % Pin (32–89%), thus they are very impacted by AMD.(A2) Agrio, And_Chorrito, Olivargas, Sancho, And_Cobica, Lagunazo, Garnacha have a higher pH range (3.27–5.8) than that of previous group, A1. However, Lagunazo, Agrio and Garnacha have higher pH (5.5–5.9), lower sulfate and lower ∑ % Pin, when compared to other sites from this group. Also due to their higher number of species (∑ Sp.), they are co-located on the rightest side of this sub-cluster.Pinnularia aljustrelica (∑ % Pin 97%) which could be responsible for its more distant disposition, which is related with the most AMD affected dams (sub-cluster A1). (A3) Notably, all samples have a very high Pearson proximity ratio, with the exception of Marismillas, which stays at a considerable Euclidean quadratic distance from the rest of the sites of sub-clusters A1 and A2. Marismillas, sticks to a separated sub-cluster, A3. It receives high quantities of dissolved and particulate matter originating from the waste rock tailings of the Rio Tinto Complex mines. Thus, water from this dam comes from the Tinto River and is AMD-affected with acid pH and high sulfate and Fe loads . Nowadays, this dam is clogged with fine sediments  Campanario, Dique I, La Joya, Chanza, Corumbel, Dique II, Herrerias with a pH range of 5.67–6.68 and the highest number of species (∑ Sp.).(B2) Puerto Leon, Silillos I, Silillos II which is distinguished from the previous group due to its lower pH range of 5.75–5.86 and a medium number of species (∑ Sp.).In this work, the dbRDA  was compdbRDA has been used previously in similar scenarios . Essenti42−, Cu, Mn, Pb on one side and EC, TDS, Cd, Co, Al, As, Ni, Zn on the other side and the pH alone, which is the opposite of the previous parameters. pH has a negative significant correlation of −0.830 with the dbRDA coordinate axis 1, separating the two groups on the left of the graph with the highest pH (4.5–7), and, the two groups of samples with the lowest pH (2–4.5) on the right of the graph. Also, SO42− and Mn show an important correlation of 0.45 with dbRDA coordinate axis 2 and are more correlated with sites with low pH: Gran, Maris, Aac, Gos, Andc.The dbRDA can be considered as a hybridization between a PCA  and a factorial analysis. This allows the visualization of the physico-chemical variables that show a higher Pearson’s correlation proximity: SOIn relation to the grouping of sampling sites, these are defined as four groups: (1) Maris, Aac, Gos, Cmora (pH 2–3); (2) Andc, San, And, Dpin (pH 3–4.5); (3) Lagu, Gran, Pleon, Oliv, Chan, SilI, SilII, Joya, Gar, Agrio, Camp, Corum (pH 4.5–6); and (4) Herr, Diq I, Diq II pH 6–7.42− (northeast quadrant) and others are more related with EC and the other metals (southeast quadrant). They represent the dams with the highest degree of AMD contamination, which were also grouped together in the cluster of sampling sites. Groups 1 and 2 seem to mix in a certain way, some sites are more related with Cu, Mn, Pb and SOdbRDA validates the previous cluster analysis, that is, pH is the most important environmental factor driving the totality of the communities. Analysis of diatom communities shows the species’ contribution to the ecological status of the water dams by considering the frequency and abundance of each taxon, and their relationship to specific physico-chemical conditions. A total of 118 species were found in the 23 water dams, however, many had a low expression (43 species with abundance < 1%). Eunotia exigua (Brebisson ex Kützing) (EEXI) Rabenhorst, Nitzschia cf. thermalis (Kützing) (NTHM), Pinnularia acidophila Hoffman and Krammer (PACI); Pinnularia acoricola Hustedt (PACO); Pinnularia subcapitata Gregory (PSCA) and Pinnularia aljustrelica Luís, Almeida et Ector (PALJ). These species have been found by other authors in similar environments that are characterized by their extreme conditions [From the four groups first in the dbRDA, the dominant diatom species in each group found by SIMPER analysis is presented in nditions ,19,20,52nditions .Achnanthidium minutissimum (Kützing) Czarnecki (ADMI), dominate together with species from the genera Navicula (NAVI) and Nitzschia (NITZ). In dams with a pH of 4.5–6, the total number of species duplicated, increasing the diversity of diatom communities. Acidophilic species do not disappear, however, the neutrophilic species appear to be dominant, for example, Nitzschia frustulum (Kütz.) Grunow var. bulnheimiana (Rabh.) Grunow (NFBU), Nitzschia amphibia Grunow (NAMP) and Achnanthidium exiguum (Grunow) Czarnecki (ADEG) started to appear abundantly. Others, less dominant, such as Navicula veneta Kützing (NVEN) and Achnanthidium minutissimum (Kützing) Czarnecki (ADMI) also appeared in this group (pH 6–7).In dams with a pH of 6–7, the total number of species duplicates is higher compared to the previous group (4.5–6), and this group has the most diverse communities. Alcaliphilic species such as This was the first time that a diatom inventory was done for AMD impacted dams in the Spanish area of the Iberian Pyrite Belt. The physico-chemical results indicate that the 23 studied dams are subject to sulfates and metallic load from AMD contaminated water courses, although at different levels. The clear response of diatom communities to physico-chemical variations in water was observed through cluster and dbRDA analysis. Pinnularia aljustrelica (97%). Through cluster analysis, it was noted that three variables: pH, ∑ % Pin and ∑ Sp. mainly conditioned the communities’ and hydrochemical behavior. Marismillas dam was alone in a separated sub-cluster, A3, because of its specific environment (AMD-contaminated waters vs. Nerva’s WWTP inputs), with communities dominated by Maris, Aac, Gos, Cmora (pH 2–3);Andc, San, And, Dpin (pH 3–4.5);Gran, Pleon, Oliv, Chan, Lagu, SilI, SilII, Joya, Gar, Agrio, Camp, Corum (pH 4.5–6);Herr, Diq I, Diq II (pH 6–7).Through dbRDA analysis, the aggrupation of diatoms (with a new variable added: % of abundance of each species and not the sum of number of species in each site (∑ Sp.)) into four pH groups was observed, in response to the intrinsic physico-chemical conditions that govern these ecosystems: The groups of dams were coincident in both cluster and dbRDa analysis, with the exception of Agrio, Garnacha and Lagunazo. These three dams have pH > 4.5 and in dbRDa they were put in the corresponding groups, (pH > 4.5), but in cluster analysis, they were put together with sites of pH < 4.5, due to their higher number of species (∑ Sp.), a variable not included in the dbRDA analysis. Pinnularia aljustrelica, Pinnularia acidophila, Pinnularia acoricola and Eunotia exigua) validates the importance of diatoms as bioindicators of AMD affected dams, contributing to the pH, as well as to the grouping of dams given the diversity of the hydrochemical patterns observed.In summary, each dam presents different degrees of contamination and the existence of a global pattern behavior is not clear. Without rainfall events, it is undoubtedly the pH that controls the metal and sulfate dissolution, which in turn regulates the conductivity values. This absence of a clear global pattern for all of the dams can be interpreted as a consequence of the coexistence of very distinct mineral paragenesis throughout the IPB, as well as the diversity in the size and nature of the watersheds. Other factors, such as the intensity and duration of the mining processes also contribute to the development of individual hydrochemical patterns. Species’ growth and their specific tolerance for distinct pH ranges, especially those tolerant to low pH (e.g.,


In this cohort, the milk microbiome composition seemed to change following the infant’s latching to the mother’s breast, shifting toward a more diverse microbial community dominated by typical oral microbes, i.e., Streptococcus and Rothia. Even if all infants in the present study were fed human milk, features typical of healthy, full term, exclusively breastfed infants, i.e., high percentages of Bifidobacterium and low abundances of Pseudomonas in fecal and oral samples, respectively, were detected in samples taken after actual breastfeeding started. These findings underline the importance of encouraging not only human milk feeding, but also an early start of actual breastfeeding in preterm infants, since the infant’s latching to the mother’s breast might constitute an independent factor helping the health-promoting assembly of the infant gut microbiome.Mother’s own milk represents the optimal source for preterm infant nutrition, as it promotes immune defenses and gastrointestinal function, protects against necrotizing enterocolitis, improves long-term clinical outcome and is hypothesized to drive gut microbiota assembly. Preterm infants at birth usually do not receive their mother’s milk directly from the breast, because active suckling and coordination between suckling, swallowing and breathing do not develop until 32–34 weeks gestational age, but actual breastfeeding is usually possible as they grow older. Here, we enrolled moderately preterm infants  to longitudinally characterize mothers’ milk and infants’ gut and oral microbiomes, up to more than 200 days after birth, through 16S rRNA sequencing. This peculiar population offers the chance to disentangle the differential contribution of human milk feeding  The microbial community that colonizes the neonatal body in early life has been acknowledged as one of the main contributors to short and long-term infant health status , and thuThe establishment of the infant gut microbial community is a topic of great interest for both scientific and clinical communities. The impact of many covariates on the composition and establishment of the infant microbiome has been thoroughly explored, pointing out that gestational age, mode of delivery, and type of nutrition  could be among the variables with the greatest impact . Among tper se vs. latching and actual breastfeeding in the development of the infant gut microbiota, moderately preterm infants  were recruited in the present study, and mothers’ milk and infants’ gut and oral microbiomes were longitudinally characterized, before and after the beginning of actual breastfeeding, up to 210 days after birth.Preterm infants at birth usually do not receive their mother’s milk directly from the breast, because active suckling and coordination between suckling, swallowing and breathing are not developed until 32–34 weeks postmenstrual age . However1) and receiving any human milk feeding (MOM and/or DHM). Exclusive formula feeding was considered as an exclusion criterion. Demographic and clinical data were recorded in a specific case report form and summarized in Table ad hoc containers, in accordance with the guidelines for human milk collection used in human milk banks (Sixteen mother-infant pairs (16 mothers and 21 infants) were recruited at the Neonatal Intensive Care Unit of S. Orsola-Malpighi Hospital in Bologna, Italy. Among the recruited infants, there were 11 singletons and 5 twin pairs (1 monochorionic monoamniotic and 4 monochorionic biamniotic). Infants were recruited if moderately preterm . Samples were then treated according to the DNeasy Blood & Tissue kit (QIAGEN) instructions, following the protocol for Gram positive bacteria  with a modified protocol . Brieflybacteria . All ext2.For each sample, the V3–V4 region of the 16S rRNA gene was PCR amplified in 25 μl final volume containing 2.5 μl of microbial DNA , 2X KAPA HiFi HotStart ReadyMix , and 200 nM of S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21 primers  carrying3, implemented with the libraries vegan, gplots, PMCMR, and made4. Filtering of bacterial taxa was performed by keeping the genera and families showing a minimum relative abundance of 0.2% and 1%, respectively, in at least 4% of the considered samples. Beta diversity was estimated by computing Bray-Curtis distances between genus-level relative abundance profiles. Bray-Curtis distances were used for Principal Coordinates Analysis (PCoA), and the significance of separation was tested by permutational multivariate analysis of variance using the function “Adonis” of the vegan package. Bacterial groups with the largest contribution to the ordination space were found by using the function envfit of the R package vegan on the genus relative abundances. As inspired by post-hoc test. P-values were corrected for multiple comparisons using the Benjamini-Hochberg method. A false discovery rate (FDR) of 5% was used. For comparison, data on the fecal and oral ecosystems of healthy, exclusively breastfed, full term 21-day-old infants, were retrieved from Data analysis was performed using a pipeline combining PANDAseq (paired-end assembler for Illumina sequences)  version Sixteen mother-infant pairs were included in the study. The pre-defined sampling of feces and oral swabs  was completed for all infants. For five mother-infant pairs, sampling continued up to 6 or 7 months after birth  . On the contrary, the fecal ecosystem showed a tendency to being impacted by the early administration of antibiotics at days 7 and 14 , but this effect was no longer detectable at day 30 . However, at day 7 and 14 the large majority of infants taking antibiotics were also hospitalized  and not breastfed , pointing out that the separate impact of these covariates, i.e., antibiotic administration, breastfeeding and hospitalization, on the microbiota composition of preterm infants could be hard to discriminate.According to the Bray-Curtis distance PCoA performed on genus-level microbiota profiles, the structure of both oral and fecal ecosystems tended to converge toward a common configuration over time, whereas higher sample dispersion was noticeable for earlier time points. According to our multivariate analysis, features of the infant microbiota composition which correlated with postnatal age were high abundances of bacteria assigned to the genus  Figures , S5. Suc Figures . Since 1Meconia were available for 19 out of 21 infants. Confirming previous studies , the micP = 0.001 and R2 = 0.18 for oral microbiota, P = 0.001 and R2 = 0.10 for MOM microbiota). Conversely, the effect of latching on the infant’s fecal microbiota was not significant  or mixed feeding (human milk plus a certain amount of formula); indeed, only 3 fecal samples and 3 oral samples from 2 subjects (twins c13a and c13b) were taken during exclusive formula feeding       Figure  and the ) Figure . Also, M) Figure , whereas) Figure . MCT3 wa Figures . Specifi Figures .P = 0.02 and R2 = 0.07)  and showed higher dispersion on the PCoA space  were excluded because their MOM samples were not available. According to a Bray-Curtis PCoA, infant’s fecal samples taken when the MOM harbored a MCT2, mostly including earlier time points Figure , were siStaphylococcus , a feature that was already detected in the corresponding milk samples , mimicking what already observed in the milk itself  that were highly shared among the three types of samples  taken from each dyad/triad mother and infant(s). In particular, OTU518072-S. epidermidis was detected at relative abundance > 0.1% in at least 1 time point in each of 3 type of samples for 100% of the enrolled infants , which was the MCT most associated with later samples (72.3 ± 63.5 days after birth) and actual breastfeeding (100%)  in all oral samples taken during the first 4 days of life  Figure . It is w) Figure . AccordiBreast milk represents the optimal source of infant nutrition. For preterm infants, exclusive human milk feeding has been linked to improved immune defenses and gastrointestinal function, protection against serious diseases such as necrotizing enterocolitis, and improved long-term clinical outcome . It is wIn order to examine the specific role of latching and actual breastfeeding in shaping MOM and infant’s oral and gut microbial communities, we included into this study a peculiar population of preterm infants and their mothers. Moderately preterm infants, who are born between 32 and 34 weeks of gestational age, are usually unable to actively suck in the first days/weeks of life , thus inStaphylococcus  2, based on the genus-level microbiota clustering of milk samples) was more frequently retrieved in the first month after delivery and in mothers who did not breastfeed directly. On the contrary, milk samples taken after latching harbored more diverse microbial communities (that we identified as MCT1 and MCT3). In particular, a microbial community dominated by Streptococcus and with significantly higher relative abundance of Rothia (suggested MCT3), which are both typical oral microbes in children (In breastfeeding mothers, the composition of the milk microbiome is known to be relatively stable over time . Howeverchildren , was assBifidobacterium in the infant’s gut, with a median relative abundance that was similar to that obtained by our research group in a previous study on healthy, exclusively breastfed, full term infants (The human milk microbiome is hypothesized to seed the infant gut , but lit infants . The gut infants , in modePseudomonas was observed in oral samples taken in correspondence to milk samples harboring MCT3, and it was similar to that measured in the mouth of the full term infants from our previous study (Pseudomonas can be an oral and tracheal pathogen found in preterm infants, particularly associated with intubation practices and antibiotics (Streptococcus, a well-known signature of oral ecosystem maturation exerting an inhibitory activity on Pseudomonas growth (Pseudomonas has also been strongly associated with skin-to-skin care in preterm infants (Observation analogous to those made on the fecal microbiota was made on the oral ecosystem of moderately preterm infants: the lowest relative abundance of us study . Pseudomibiotics . It is as growth , was the infants . Taken tPB, AZ, GF, and LC conceived the study. AA applied for Ethical Committee approval. SQ, EB, and IB performed the experimental investigation. EB, SR, and MS analyzed bioinformatics and statistics of this work. EB, SQ, and AA wrote the original draft of the manuscript. EB, MC, AA, and ST wrote, reviewed, and edited the manuscript. AA, IB, and SQ collected the samples and curated the data. EB and MS prepared the figures. PB, LC, MC, and EB acquired funding. All authors discussed the results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This review presents an survey to the biological importance of sesquiterpene lactones (SLs) in the fight against four infectious neglected tropical diseases (NTDs)—leishmaniasis, schistosomiasis, Chagas disease, and sleeping sickness—as alternatives to the current chemotherapies that display several problems such as low effectiveness, resistance, and high toxicity. Several studies have demonstrated the great potential of some SLs as therapeutic agents for these NTDs and the relationship between the protozoal activities with their chemical structure. Recently, Computer-Aided Drug Design (CADD) studies have helped increase the knowledge of SLs regarding their mechanisms, the discovery of new lead molecules, the identification of pharmacophore groups and increase the biological activity by employing in silico tools such as molecular docking, virtual screening and Quantitative-Structure Activity Relationship (QSAR) studies. Neglected tropical diseases (NTDs) are diseases that are present around the world in tropical and subtropical regions. They affect about one billion people, who commonly live in poverty , from 15According to the classification of the World Health Organization, 19 NTDs exist , of whicTrypanosoma cruzi, present many side effects in patients. The low effectivity in the chronic phase of the disease, the high risk of death from melarsoprol, an arsenic derivative for treating the chronic phase in human African trypanosomiasis, and the increase in cases of resistance of schistosoma to praziquantel treatment are some examples of chemotherapy actually used for NTDs that present several problems and prevent the control and elimination of these diseases.Antimonial compounds are the main treatment against leishmaniasis, which presents high toxicity and resistance in some endemic regions. Nifurtimox and benznidazole, the drugs employed against Because of this, it is necessary to discover new low cost drugs that are highly effective with low toxicity. Humans have thus turned to plants as a source of compounds for the treatment of many diseases, and in addition to being an option to find solutions for many health problems of most of the world’s population, is an important industry, as plants generate over $25 billion dollars per year in value based only on the relevant pharmaceuticals derived from plants .Plasmodium falciparum, opened the panorama with respect to research into new therapeutics for leishmaniasis, trypanosomiasis, and schistosomiasis from this type of secondary metabolite, and in addition to the antimalarial activity, the drug has antitumor potential and shows prevention of neurodegeneration, antimigraine activity, analgesic, and sedative activities santamarin  [Interaction of the guaianolides and eudesmanolides was stronger with olin 81,  with Lmaarin 82,  exhibiti kJ/mol) .T. brucei, T. rhodesiense, T. cruzi, and L. donovani—as well as Plasmodium falciparum and the cytotoxic activity against L6 rat skeletal myoblast cells. QSAR studies were performed with those data using molecular modeling and multivariate data analysis tools to evaluate the variance in the biological activity in terms of the relative influences of various molecular descriptors. Helenalin  against four protozoan pathogens—alin 30,  and its 0, 83–85  show higxide 86, , a xanthdonovani .T. brucei and L6-cytotoxicity is studied through the variation in the coefficients of determination (r2) obtained by multiple linear regression. The whole molecule set generated low r2 values for T. brucei and L6-cytotoxicity , and the significance of these two descriptors was confirmed by F- and t-tests. While only considering 16 related compounds of the helenanolides 30, 32, 50, 51, 83–85, and 87–95 series, higher r2 values against T. brucei and L6-cytotoxicity  were achieved. The authors interpreted that in the case of compounds possessing very similar molecular structure, the modulating influence of other structural factors on bioactivity is small compared with the major impact of chemical reactivity  [The influence of presence or absence of α-methylene-γ-lactone and α,β-unsaturated ketone in activity against  groups) ,88,89.T. brucei activity). The final model contained three latent variables from 20 selected descriptors and presented a Q2 value for T. brucei and L6 cells of 0.71 (for both), which is significantly higher than the model obtained using the whole set of molecules and seven latent variables from 44 descriptors that presented a Q2 value of 0.52 and 0.41, respectively. However, PLS models only were evaluated through internal validation  without any external test [Three QSAR models was evaluated. The AM1 semi-empirical method was initially used to optimize the molecule structure for posteriorly calculating 44 descriptors in the MOE software to generate an initial model. PLS analysis was performed using two dependent variables  was also studied computationally through molecular docking using two sirtuins (silent-information regulator 2 proteins), T. cruzi Sir2RP1, and Sir2RP3, which act as antiparasitic targets. The PLP score was used to rank and evaluate the docking poses and these were compared to select inhibitors of sirtuin classes I and III, AGK2, and thiobarbiturate 6. PLP docking scores obtained were 40.6 (Sir2RP1) and 49.2 (Sir2RP3), and intermediate values with respect to the best scores were reached by anacardic acid  of 80.7 for Sir2RP1 and aculeatin D of 90.3 for Sir2RP3 [Helenalin  and refined by the semi-empirical AM1 method. A set of one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) descriptors was calculated through PaDEL 2.11. Additionally, Weighted Holistic Invariant Molecular (WHIM) 3D molecular descriptors used  coordinates of a molecule were used within different weighting schemes to generate a value that represents its structural features with respect to size, shape, symmetry, and atom distribution [2r. SLs studied were divided in two groups, a training group (composed by 12 SLs) and a test group  whose selection method was not reported. pIC50 values predicted by the best QSAR model (2r = 0.99), which relates two descriptors, LAC  and Wvol (a holistic descriptor related to steric factors), are close to the experimental values reported for these 3 SLs. Cross-validation coefficient values are not reported. The authors found that a correlation of the observed antitrypanosomal activity existed with the steric parameters of the set of molecules tested and concluded that SLs have a high potential as lead molecules against Chagas disease [A purely explanatory QSAR study was performed for 15 sesquiterpene lactones ribution . The QSA disease .T. brucei rhodiense, furanoheliangolide-type compounds appear as interesting leads against human African trypanosomiasis. From the three-dimensional structures of the 69 molecules, whose energies were minimized through AM1 semi-empirical method, 123 molecular descriptors were calculated. The whole set of molecules was divided into a training set (46 molecules) and test set (23 molecules), and this division was based on a structural diversity classification. A QSAR model generated, which is correlated with the presence of reactive structure elements and their electronic properties, is important to the hydrophobic volume balance, and is codified by VolSurf descriptors with a significant contribution in the model [2 value [An excellent example of the discovery of new compounds with high antiparasitic activity from in silico researches was found from the prediction of in vitro activity of 1750 SLs structures of a virtual library through a QSAR model performed with 69 molecules. Because of the high levels of activity against he model . Interna[2 value .T. brucei rhodesiense, 71 molecules presented pIC50 values greater than or equal to 7.0, and 15 compounds corresponded to furanoheliangolide-type SLs. Therefore, the antitrypanosomial activities of four of these were evaluated, goyazensolide , budlein A , and 4,5-dihydro-2′,3′-epoxy-15-deoxygoyazensolide  and 4,15-isoatripicolide tiglate , and experimental pIC50 values of 7.14, 7.14, 6.70, and 7.82, respectively, were obtained. The QSAR model underestimated the activities of budlein A and 4,15-isoatripicolide tiglate, while it overestimated goyazensolide [In the prediction of activity against ide 107, , which p108, T. brucei rhodesiense, with a IC50 value of 15 nM and a selectivity index of 77 , which is an interesting lead against T. brucei rhodesiense [From this QSAR model and subsequent validation through in vitro tests performed, furanoheliangolide 108,  4,15-isodesiense .Trypanosoma in VirtualToxLab, an in silico tool for estimating the toxic potential of drugs, chemicals, and natural products. The results obtained showed a wide range of potential toxicity, from 0.161 classified as benign of germacranolide  with activity against ide 109,  to 0.5930 family .In this review, we have covered the problematic aspects of the chemotherapies currently used against leishmaniasis, Chagas disease (American trypanosomiasis), sleeping sickness (human African trypanosomiasis), and schistosomiasis, and we showed how natural products have emerged as alternatives for the development of new medications against these NTDs. Specifically, sesquiterpene lactones, and secondary metabolites characteristic of the Asteraceae family have shown promising in vitro and in vivo activity, and some promising molecules have emerged from these studies.Recently, CADD studies (mainly molecular docking and QSAR) have appeared with the aim to obtain new molecules by modify some existing molecules and understanding the mechanism of action of the SLs, which also increases the knowledge of this topic. In vitro tests for confirming the mechanism of action of the most active selected SLs in the docking studies here referenced were not reported. Hence, the obtained information from those studies are inconclusive regarding its enzymatic activities and demand further study.This research area is still developing and an example of that is the case of schistosomiasis, where there have been no in silico studies with SLs. Therefore, it is important to continue performing CADD studies to find solutions with low cost and few side effects for the health and social problems caused by NTDs.


Mixed acinar-endocrine carcinoma (MAEC) of the pancreas is a rare neoplasm, consisting of at least 25%–30% of acinar and neuroendocrine populations. Patients are often middle-aged and present with nonspecific symptoms. Imaging typically reveals a solid lesion in the pancreatic head. Management involves surgical resection and the overall prognosis is variable. Here, we present a case of a 48-year-old male who presented with a MAEC arising from duodenal pancreatic heterotopia. This is the one of the first cases, with histologic evidence, of MAEC arising from pancreatic heterotopia. Pancreatic tumors are rare and may arise from either endocrine or exocrine components. Neuroendocrine cells make up the endocrine component, while ductal and acinar cells make up the exocrine component. The majority of pancreatic malignancies are ductal adenocarcinomas >75%), followed by neuroendocrine tumors (NET) (7%), and acinar cell carcinomas (ACC) 1%) % 1, 2]..2]. Up t%, follow..2]. Up A 48-year-old male with past medical history of asthma, chronic pain, and obesity presented with progressive fatigue and epigastric discomfort for approximately six to eight weeks. A complete blood count revealed a hemoglobin level of 5.2 g/dL and esophagogastroduodenoscopy (EGD) demonstrated a large ulcerated mass in the duodenum, occupying approximately 90% of the circumference of the duodenal wall. The mass extended from the apex of the duodenal bulb to the major papilla; however, the major papilla was unremarkable. A follow up computed tomography scan showed a 6.0 × 5.5 × 5.0 cm hypoattenuating mass involving the mesenteric aspect of the second portion of the duodenum, which approached the proximal third segment of the duodenum. Additionally, the mass appeared to involve the pancreatic head and uncinate process; however, no hepatobiliary or pancreatic ductal obstruction was noted. Biopsy revealed a poorly differentiated carcinoma with neuroendocrine features. The patient underwent a pancreaticoduodenectomy (Whipple procedure) which showed a 10.2 × 8 × 2.7 cm pink-tan, lobulated, fungating mass with central necrosis within the duodenum. The mass was centered in the lumen of the duodenum with a well-demarcated (pushing) front of macroscopic invasion into the pancreas .Microscopic examination of the lesion showed a neoplasm arranged primarily in lobules with prominent acinar formation. There were focal areas where the tumor was arranged in solid sheets. The neoplasm was located in the duodenum adjacent to an area of pancreatic heterotopia Figures . Some ofFollowing surgical resection, the postoperative course was complicated by several bacterial infections, pulmonary abscesses, and liver metastases. Unfortunately, the patient died approximately six months after initial presentation.MAEC is a rare pancreatic tumor, consisting of at least 25%–30% acinar and neuroendocrine cells . These tPatients undergo initial evaluation with fine-needle aspiration, yet cytologic evaluation of MAEC can be extremely difficult. In some circumstances, it is easy to identify cells in an acinar formation that have granular cytoplasm and prominent nucleoli, suggesting ACC . SimilarDefinitive diagnosis of MAEC relies upon examination of the resection specimen to show the necessary proportion of acinar and neuroendocrine cells. There are two main patterns of MAEC described in the literature, which should be considered when the differential includes ACC, NET, and MAEC. These patterns include 1) two morphologically distinct and isolated populations of cells or (2) two intermingled populations of cells , 10, 15. two morpThere are few reports of MAEC published in the literature, and even fewer published arising in pancreatic heterotopia. Moncur et al.  describeThe etiology and behavior of MAEC are not well understood. Literature documents fairly poor prognosis, similar to that of ACC, and suggests that tumors with increased numbers of neuroendocrine cells have a better prognosis . Some auMEN1, ATRX, DAXX, TSC2, PTEN, Rb, and TP53 [ SMAD4, JAK1, BRAF, RB1, TP53, APC, ARID1A, GNAS, MLL3, and PTEN [The molecular characteristics of pancreatic NET, neuroendocrinecarcinoma (NEC), and ACC have been well characterized. Pancreatic NET and NEC have an average of 16 somatic mutations. The major genes involved in these somatic mutations are and TP53 . ACC hasand PTEN . On the and PTEN  have desand PTEN .Since the pathogenesis is not fully understood, there is no standardized treatment available for MAEC. With comparable aggressiveness to that of ACC , 9, 10, In conclusion, we present a rare case of MAEC arising from pancreatic heterotopia in the duodenum. Owing to the morphologic overlap between MAEC, ACC, and NET, it is important to consider these differentials, especially when tissue is limited on FNA or cell block. ACC are much more aggressive than NET of the pancreas, and it is suspected that MAEC have a prognosis similar to that of ACC. Thus, it is important to accurately diagnose these lesions as the management may differ. With increased recognition of MAEC, especially with improved IHC, it is likely that additional studies will be completed which will contribute to improved management of these neoplasms.


Supplemental Digital Content is available in the text In Korea, low back pain (LBP) which is occupation-related symptom is one of the major health issues owing to rapid industrialization. Even traditional Korean medicine has the long history in pain treatment, there still has been lack of supporting evidence on herbal prescription itself. Sogyeonghwalhyeol-tang, a Korean herbal medicine prescription, has been suggested as a medication for treating chronic LBP as well as work-related pains.This study aims to evaluate the clinical efficacy and safety of herbal medicine, Sogyeonghwalhyeol-tang on work-related chronic LBP patients.This trial is designed as a multicenter, randomized, controlled, clinical trial. Seventy-two participants who have chief complaint of LBP in Korean medicine rehabilitation center will be randomly assigned to ether Sogyeonghwalhyeol-tang group or placebo group with a ratio of 1:1. They will receive assigned drugs in 4 weeks and follow-up for 2 weeks.The result of this study will provide the valuable information for efficacy and safety of Sogyeonghwalhyeol-tang for patients with work-related chronic LBP. It is caused by repetitive posture, maintaining a fixed position for prolonged duration, and rapid work pace; the resulting disability can become a socioeconomic problem and lower an individuals’ quality of life. According to the 2018 annual report by the Korea Occupational Safety and Health Agency, work-related injuries or resulting disability of low back comprise 40.5% (3281/8105) of the overall work-related diseases or injuries.,3 However, traditional Korean medicine (TKM) utilizes multiple, individualized treatment methods, such as acupuncture, moxibustion, Chuna, and various herbal medicines to treat the overall discomfort because of Wr-cLBP with a holistic approach. As per the 2017 report by the Korean Medical Use and Consumption Survey, low back pain (LBP) is a frequent reason for seeking medical care at Korean medical institution, with a lifetime utilization of 52.7%. This result indicates that a considerable number of patients with LBP approach TKM institutions to treat their discomfort in Korea.Wr-cLBP generally causes the loss of productivity, leading to economic burden, and needs long-term treatment care plan. Treatment with conventional medicine promotes health and prevents the disease to reduce and stabilize an individual's quality of their life. Conventional medical approaches generally include the use of non-pharmacologic treatments, such as physical therapy and chiropractic care, based on clinical practice guideline. Several studies have reported on pain related to arthritis and neurologic symptoms.–10Sogyeonghwalhyeol-tang (SGHH) is addressed in one of the famous traditional Chinese medicine classics, Wanbinghuichun, and is presumed to mediate its effect by activating the blood and dredging the meridians and collaterals to treat abnormal musculoskeletal symptoms, such as pain, palsy, and stiffness.Although TKM has a long history of treating Wr-cLBP, no well-designed clinical trial has been undertaken to evaluate the potential efficacy of SGHH in the Korean population. Therefore, we will examine the feasibility of a full, randomized, placebo-controlled clinical trial to evaluate the efficacy of SGHH on Korean patients with work-related LBP to provide more conclusive evidence of its therapeutic efficacy.1.1This pilot trial will be a multicenter, double-blind, randomized, parallel-group, placebo-controlled clinical trial that will be conducted in 4 university-affiliated TKM hospitals: Gil oriental medical hospital of Gachon university, Semyung University Second Affiliated oriental medical hospital at Jecheon, Dunsan Korean medicine hospital of Daejeon university, and oriental Medicine hospital of Sangji University.On confirming their participation in this trial, the recruited participants will undergo an evaluation for identifying eligible candidates. A total of 72 eligible participants will have a 7-day run-in period before randomization. After randomization, the participants will receive treatment for 4weeks, with additional 4 weeks of follow-up, during which they will visit the clinic once every 2 weeks. The flow chart describing the trial protocol is shown in Figure 1.2 hospital workers, office worker, and transit workers  will be used for convenience. Each dose packet contains 0.83 g of Paeoniae radix, 0.66 g of Angelicae radix, 0.66 g of Rehmaniae radix, 0.66 g of Atractylodis japonica Koidzumi, 0.66 g of Cnidii rhizome, 0.66 g of Persicae semen, 0.66 g of Poria, 0.50 g of Achyranthis radix, 0.50 g of Clematidis radix, 0.50 g of Sinomeni caulis et rhizome, 0.50 g of Notopterygii rhizome, 0.50 g of Saposhnikoviae radix, 0.50 g of Gentianae scabrae radix, 0.50 g of Aurantii nobilis pericarpium, 0.33 g of Angelicae dahuricae radix, 0.33 g of Glycyrrhizae radix, and 0.33 g of Zingiberis rhizome. Moreover, the placebo will be manufactured relatively similar in appearance to SGHH, under strict quality-control system such that the participants cannot distinguish between the 2. Placebo contains 5.1 g of lactose, 3.0 g of cornstarch, and 0.3 g of Ssanghwa herbal flavor.To improve adherence of investigational drug, we will ask participants to return the unused drugs for drug compliance at every study visit.1.4 functional status by the Korean version of the Roland Morris Disability Questionnaire (RMDQ), and quality of life by European Quality of life 5 Dimension (EQ5D).,19 All questionnaires will be completed by the participants on their own at each follow-up visit.To evaluate the intensity of the LBP, we have selected 3 assessment tools such as: assessing pain intensity by numeric rating scale (NRS),1.51.5.1The 10-point numerical scale is used to assess the intensity of LBP on a scale of 0 (no pain) to 10 (worst pain). The participants will be asked to mark their pain intensity on the scale at each visit. The change in NRS score for pain intensity between the 2 groups will be analyzed.1.61.6.1The Korean version of RMDQ, the most responsive questionnaire that reflects the gradual clinical changes, will be used to evaluate the disability of daily life in this study. It consists of 24 items representing general physical functions that are likely to be affected by LBP. Higher numbers on the questionnaire's scale reflect greater levels of disability.1.6.2The Korean version of EQ-5D is a validated measure for assessing the health-related quality of life and consists of 2 types of measures—the descriptive system of EQ-5D and VAS of EQ (EQ-VAS). The descriptive system comprises 5 dimensions, including mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, and each dimension includes 3 levels of severity of discomfort to daily life. The EQ-VAS is used as a quantitative measurement of health status self-reported by the participants.1.6.3 BUN, creatinine, ALT, AST, and γ-GT are additionally assessed at visit number 3, and moreover, the vital signs will be checked at every visit.Safety assessment, including physical examination, complete blood count test, blood biochemistry, renal function test , and liver function test  will be conducted during screening visit and at visit number 4.1.7This trial is a pilot study to examine the feasibility of a full randomized controlled trial for evaluating the efficacy of SGHH and to determine the effect size for further large-scale studies. A sample cohort comprising 72 patients has been considered after assessing patients’ frequency of clinic visits, availability of study staff, amount of funding, and dropout rates at each participating site.1.8Participant recruitment will take place in 4 university affiliated Korean medicine hospitals. Each site may include participants by means of website, hospital bulletin board, and local community-based newspaper publishing recruitment posters.1.9The eligible participant will be randomly assigned to either the treatment (SGHH) or placebo group with an allocation ratio of 1:1. The random assignment will be done by the web-based service provided by the contract research organization (CRO), Institute of Safety and Effectiveness Evaluation for Korean Medicine (ISEE). The random sequence will be generated by an independent study staff from ISEE using the SAS 6.1 program, and stratified by the participating institutions using block sizes of 2 and 4.Once the participant is randomly assigned to a group, either the participant or the investigator will be made aware of the type of intervention the participant will receive until the end of the study period. The interventional agents will be packaged similarly such that they are indistinguishable by appearance, and distributed by independent study site-assigned pharmacists or assistants who will be blinded to the random allocation. In case of withdrawal symptoms or occurrence of urgent medical conditions, the site's principal investigator will directly inform the CRO and sponsor to unblind the participant's intervention according to the standard operating procedures (SOPs).1.10t test for continuous data and χ2 or Fisher exact tests for categorical data will be used. The repeated measures analysis of variance will be used to compare the baseline measurements with visit number 4, and follow-up numbers 1 and 2. Comparison of means of the continuous variables  will be expressed as mean differences with 95% confidence intervals (CIs). Missing data will be adjusted using the last-observation carried-forward imputation method. Statistical analyses will be performed using SPSS 23.0 program and P < .05 will be considered statistically significant. There will be no interim analysis unless the pattern identification decide to stop the trial and access authority to the final dataset will be only given to the pattern identification.Data analysis will be conducted by both intent-to-treat (ITT) and per-protocol (PP) analysis by an independent statistics expert, blinded to the study. Independent sample 1.11The monitoring of collected data and research performances will be conducted by ISEE on a regular basis. The monitoring will begin when the first enrolled participant completes the required number of visits. All the participating sites will be monitored based on the SOPs during the trial progress. Auditing is not scheduled for this pilot study. Data validation and verification, such as double data entry and range checks for data values, will be performed for ensuring data quality. Any other committee, such as a coordinating center, a steering committee, an endpoint adjudication committee, among others, will not be applicable in the present study. No auditing will be performed as well.1.12All participants will be informed of potential adverse events (AEs) occurring during the trial and cases of AE will be reported to the site trial staff immediately. Each AE will be recorded in the electronic medical record and case report form by the site trial staff and all AEs will be assessed for causality. If serious adverse events (SAEs) occur, the site staff will report to the site principle investigator and institutional review board (IRB) at the earliest. After receiving an SAE report, the site principle investigator will decide on the continuation of the participant in the trial.1.13Participants may withdraw from the trial at any time, without penalty. If violation of eligibility criteria or situations, such as concomitant drugs, low drug compliance occurs, principle investigator decide the termination of the study. All the progress notes on withdrawal and dropouts are recorded on the CRFs with reasons.1.14stored in locked cabinets in secured areas to protect confidentiality. When important protocol changes occur, the changes must be approved by the IRBs before implementation. The results of this study will be published in a scientific journal.This study has been designed in accordance with the amended Declaration of Helsinki and the regulations of the “Good Clinical Practice” principles of the Korea Food and Drug Administration. It has been approved by the IRB of the 4 participating hospitals . The current version of the protocol is 1.1 and it has been formulated according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Statement (see Supplementary Appendix 3). All items from the World Health Organization Trial Registration Data Set have been drawn. All participants will receive detailed explanation and sufficient time to determine trial participation, and hand-written informed consent will be obtained from all participants before proceeding with any of the trial procedures. All personal information will be 2Wr-cLBP is a common disease affecting the patients’ overall quality of life in industrial countries. In this study, we will explore the efficacy and safety of the herbal medicine extract, SGHH, in patients with work-related LBP. To our best knowledge, this is the first randomized controlled trial that will evaluate the efficacy of an herbal medicinal extract as a single intervention in cases of Wr-cLBP.There are several limitations to this trial. First, because of the limited number of participating institutions and considering their possible enrollment conditions, we have restricted the type of occupations that will be targeted for participant enrollment. Second, owing to the limited research grant for the pilot study, our sample size is limited, and the study results cannot be generalized. However, attempts will be made to control or reduce any potential biases that may affect the trial results.Nevertheless, this trial will provide valuable information, such as study design, outcome variables, pain characteristics of each occupation, and statistical power for sample size calculation for future large-scale trials, and the future development of general clinical guidelines for Wr-cLBP in the field of Korean medicine.Conceptualization: Min-Seok Oh, Sun Joong Kim, Yun-Yeop Cha, Eun Jung Lee, Yunkyung Song.Methodology: Bo-Hyoung Jang, Min-Seok Oh, Sun Joong Kim, Yun-Yeop Cha, Yunkyung Song.Project administration: Yunkyung Song, Seung-Gyu Ko.Supervision: Yunkyung Song, Seung-Gyu Ko.Writing – original draft: Youme Ko.Writing – review & editing: Bo-Hyoung Jang, Min-Seok Oh, Sun Joong Kim, Yun-Yeop Cha, Yunkyung Song.


Persistent impairment in cognitive functioning postoperatively is reported by clinical and animal studies, and is labeled as postoperative cognitive dysfunction (POCD). Evidence points to an exaggerated neuroinflammatory response resulting from peripheral systemic inflammation after surgery, with subsequent cytokine-induced glutamatergic excitotoxicity and synaptic impairment. These immunological changes, among many others, are also observed in Alzheimer’s disease. Memantine is an N-methyl-D-aspartate receptor (NMDAR) antagonist commonly used to treat Alzheimer’s disease. Surprisingly, little research exists on the role of memantine in preventing POCD. The purpose of this study is to investigate the effects of memantine on a spectrum of cognitive functions postoperatively. Mice were divided into 3 groups and each received treatment for 4 weeks. Placebo groups received a placebo then underwent either a sham procedure or a laparotomy procedure. The memantine group received memantine hydrochloride then underwent a laparotomy procedure. Cognitive tests were performed on postoperative days (POD) 1 and 7. Compared to sham-operated mice, placebo groups that underwent a laparotomy procedure showed impaired memory in the Morris water maze test, higher anxiety-like behavior in the open field and the elevated plus maze tests, increased depression-like behavior in the tail suspension test, and lack of preference for social novelty in the three-chamber test. On the other hand, memantine-treated mice that underwent a laparotomy procedure showed enhanced memory on POD7, improved depression-like behavior on POD1 and POD7, enhanced preference for social novelty on POD1, and no improvement in anxiety-like behavior. These findings suggest a potential protective effect of memantine in mice postoperatively on memory, depression-like behavior, and preference for social novelty. The allegations and remarks raised by family and friends of patients that underwent surgery, such as “He’s never been the same since his operation” or “She’s lost all interest in the family since …”, have led Bedford  to publiTo distinguish it from postoperative delirium, which occurs in the first few days following surgery, POCD is defined as persistent cognitive impairments (lasting for more than one week) after surgery . Also, tThe pathophysiology underlying POCD is not yet fully understood. Accumulating evidence point to the role of peripheral systemic inflammation  in causing exaggerated inflammatory responses in the brain , as showCurrently, there is no adequate treatment or prophylaxis for POCD ,15, and Male BALB/C mice (n = 60), aged 4–6 months were used. The animals weighed 25–36 g at the beginning of the experiment. All animals were housed individually in cages of 52 cm in length × 30 cm in width × 21 cm in height, with soft bedding. The animal colony was maintained at 22 ± 2 °C and a relative humidity of 40–60%, on a 12-hour light/dark cycle (lights on at 6:00 A.M). All mice were allowed ad libitum access to laboratory chow and tap water, and were given at least 1 week to acclimate to colony conditions before experimentation began.The animals were randomly divided into three groups . PlaceboAll experimental procedures and behavioral tests in the project (55-PI-02/16) were approved by the Research Ethics Committee, the Arabian Gulf University, Manama, Bahrain. All efforts were made to minimize the number of animals used, and the animals were handled in accordance with good animal practice.For the memantine group (M), memantine hydrochloride  was administered daily for 4 weeks, as a solution in physiological saline via oral gavage, at a therapeutic dose of 30 mg/kg/day. A steady-state plasma drug level of memantine of around 1 µmol/L in mice is considered therapeutic ,36,37, aLaparotomies and sham surgeries were performed using aseptic procedures under general anesthesia with intraperitoneal injections of ketamine  at a dose of 1.0 mg/10 g body weight and xylazine  at a dose of 0.1 mg/10 g body weight, following a previously described method .After the induction of anesthesia, the abdominal region was shaved and thoroughly cleaned with 70% ethanol  and surgical scrub . Approximately 0.5 cm below the left rib in the left upper abdominal quadrant, a 1.5 cm vertical incision was made, penetrating the skin and the peritoneal cavity. A sterile probe was inserted into the opening and manipulated the viscera and musculature for 1 min. Sterile dissolvable sutures  were used to suture the abdominal muscles. The skin was closed with silk thread sutures. To reduce the risk of infection, the wound was dressed with Polysporin (Pfizer). Sham-operated mice (PA group) were anesthetized, and the abdominal area was shaved and cleaned as described above. They remained on ketamine and xylazine for the same amount of time as their surgical counterpart (about 10 min). Upon recovery from anesthesia, all animals received buprenorphine  at a dose of 1.0 mg/10 g body weight subcutaneously for analgesia. All procedures were performed by one person to limit variability. In the postoperative period, surgical wounds were inspected daily for wound dehiscence or discharge.Behavioral tests were conducted between 6:00 A.M and 6:00 P.M. All sessions were videotaped, and were conducted and scored blindly. To eliminate possible inter-observer variability, each parameter was scored by one observer for all mice.Each animal underwent 5 training trials per day for the first day in the Morris water maze, followed by 3 trials in the second day, and a final probe trial on the third day, as previously described ,40.The maze consisted of a large circular swimming pool, measuring 140 cm in diameter and 50 cm in height, filled to a depth of 30 cm, and the water was maintained warm at 24 °C. The pool was placed in a darkened room that is illuminated by sparse red light, with surrounding extra-maze visual cues. Two imaginary diagonal lines divided the pool into 4 equal quadrants. Mice were given acquisition trials to learn the position of a hidden platform (diameter 12 cm), submerged 2 cm below the water surface, in the center of one quadrant. Performance in the trials was averaged to yield one single data point per mice per day. On each trial, the mice were released into the water facing the wall of the pool from one of four randomly assigned positions on the perimeter of the pool , and were allowed a maximum of 120 seconds per session to find the hidden platform. They were allowed to remain on the platform for 30 seconds in between sessions. If an animal failed to find the platform within the allowed time, an experimenter would gently guide the animal to the platform.The position and movement of the animals in the pool were recorded and analyzed using the ANY-maze video-tracking system . Outcome measures were time spent to reach the platform (latency time), distance swum to reach the platform, and speed of swimming. On the third day, a final probe trial was conducted in which the platform was removed and each animal was allowed to swim for 60 seconds. The time spent in the zone that previously contained the platform was analyzed, and the selective search strategy was indicated if animals performed significantly above chance (25%).Locomotor function and anxiety were assessed using the open field test, as described previously . A squarOutcome measures were distance traveled (indicated by number of line crossings), frequency of central zone entry, and time spent in the central zone . Distance traveled reflected the general motor function. Time spent in the central zone and frequency of central area entry were measures of anxiety. At the end of each trial, the arena was cleaned using 70% ethanol to prevent olfactory cue bias.Anxiety-like behavior was assessed using the elevated plus-maze test, as described previously . The appMeasures recorded were the number of entries into an arm, and the time spent in open and closed arms. Percentage of entries into open arms, time spent in open arms, and total number of entries were analyzed. Entry into an arm was defined as all four limbs in an arm. Anxiety-like behavior was indicated by decreased number of entries into the open arms, and spending less time in the open arms. Total number of entries into open and closed arms was a measure of general activity. After each session, the apparatus was cleaned thoroughly with 70% ethanol.The tail-suspension test was used to assess depression-related behavior, as described previously . A rectaThe time that each mouse spent as immobile was measured. Mobility included escape-related behaviors, such as trying to reach the walls of the apparatus and the suspension bar, strong shaking of the body, and movement of the limbs akin to running. Small movements that were confined to the front legs but without the involvement of the hind legs were not counted as mobility. Swinging movements resulting from previous bouts of mobility also were not counted as mobility. Lack of escape-related behavior is considered immobility, which is indicative of depression-like behavior.The three-chamber test  was usedIn session 1, an unfamiliar mouse (stranger 1) was placed inside the wire cage that was located in one of the side chambers. The placement of stranger 1 in the left or right side of the chamber was systematically altered between trials. The partitioning walls between the chambers were removed to allow free access for the subject mouse to explore each of the three chambers. Session 1 was recorded and continued for 10 min. The parameters assessed were time spent in each chamber and the total number of chamber entries.After the end of session 1, session 2 started immediately with the placement of a second unfamiliar mouse (stranger 2) inside the other wire cage that was placed in the center of the opposite side chamber (which was previously empty during session 1). The subject mouse had the freedom to access all three chambers. The duration for session 2 was 10 min, and the same parameters were assessed as in session 1. After each session, all chambers were cleaned with 70% ethanol to prevent olfactory cue bias.Normal sociability/social motivation (tested in session 1) was indicated by spending more time in the chamber containing stranger 1 compared to the chamber with empty cage. Intact social memory and predilection for novel experiences (tested in session 2) were indicated by spending more time in the chamber containing the newly encountered mouse (stranger 2) compared to the chamber containing the first, already-investigated mouse (stranger 1).p value of less than 0.05.Statistical analyses were performed using SPSS package (version PASW Statistic 18.0.3) and Microsoft Excel (version 15.41). All data are represented as mean ± standard error of the mean (SEM). Differences in the performance between and within groups were assessed using a one-way analysis of variance (ANOVA), followed by paired or unpaired post-hoc t-tests. Statistical significance was set at a Weight changes were recorded for all animals . No signMemory and learning were assessed using the Morris water maze. On postoperative day 1 , sham-opOn postoperative day 7 , sham-opLocomotion and anxiety-like behavior were assessed using the open field test and the elevated plus-maze. In the open field test on postoperative day 1 , no signIn the elevated plus-maze on postoperative day 1 , mice inDepression-like behavior was assessed using the tail-suspension test. On postoperative days 1 A and 7 B, placebIn the three-chamber social apparatus, normal levels of sociability were observed on postoperative day 1 , as miceOn postoperative day 7 , normal The purpose of this study is to examine the effect of memantine on a spectrum of cognitive functions postoperatively. Hovens and colleagues  point toWith regards to body weight, mice that underwent surgery (PB and M groups) suffered from weight loss on postoperative days 1 and 3, but regained their weight on postoperative day 7. Weight loss in the first few days after surgery in POCD models is commonly reported ,49, and Learning and memory were assessed using the Morris water maze test. On postoperative days 1 and 7, mice that underwent surgery (PB-POD1 and PB-POD7) spent more time and travelled more distance to reach the platform, and spent less time in the disc zone in the probe test, compared to the groups that received anesthesia and analgesia without surgery (PA-POD1 and PA-POD7). Motor function was not affected, as all groups had similar speed of swimming. These findings suggest impaired learning and memory in mice following surgery. Several studies reported similar findings ,50. The The abdominal surgery performed on mice in this study is considered minor surgery. The surgical model used  was showAnxiety may inhibit exploratory behavior in mice , as wellDecreased social activity following surgery has been reported previously by human studies ,63, and Depression was assessed using the tail-suspension test. Mice that underwent surgery (PB-POD1 and PB-POD7) showed depression-like behavior on postoperative days 1 and 7, as they had higher immobility times compared to their non-surgical counterparts (PA-POD1 and PA-POD7). Animal models of peripheral inflammation reported depression-like behavior, and attributed the phenomenon to a high turnover rate of brain serotonin as a result of an exaggerated elevation in inflammatory mediators in the brain ,67. On tAs mentioned, the pathophysiology underlying POCD is not yet fully understood, and several etiological factors have been proposed. Hypocarbia due to hyperventilation during anesthesia was shown to prolong cognitive dysfunction after surgery , but othConsistent evidence exists only for the role of postoperative peripheral inflammation in causing exaggerated neuroinflammatory responses in the brain, that manifest as cognitive decline. Severe systemic inflammation, as a result of trauma or infection, is known to affect the central nervous system (CNS) ,89,90, aInterestingly, these neuroinflammatory responses also lead to neurodegenerative changes, such as tau phosphorylation, cytokine-induced glutamatergic excitotoxicity, synaptic impairment, and most importantly, beta-amyloid accumulation, which are also characteristic of Alzheimer’s disease ,101,102.Management of POCD is centered around preventive measures. It involves identifying those who are at risk, and optimizing perioperative physical and mental health. Several measures have been shown to reduce the incidence of POCD ,106,107.However, no effective pharmacological treatment exists. Research in this area focused mostly on anti-inflammatory medications, since neuroinflammation contributes to POCD significantly. For example, the administration of small doses of ketamine, which has an anti-inflammatory effect, was shown to reduce the incidence of POCD . Minocyc2+ inflow through ion channels, which results in pathological over-excitation, and ultimately neuronal death. Furthermore, hyper-activation of NMDARs has also been associated with tau toxicity [2+ ion channel, and thus it targets both of the pathological processes that are known to be involved in POCD. Previous research also showed that memantine reduces neuroinflamamtion in animal models and improves cognitive function [In the present study, we hypothesized that memantine, an NMDAR antagonist, that is shown to be effective in treating Alzheimer’s disease ,32,33,34toxicity . As menttoxicity . Memantifunction ,113,114.function  showed tThe findings of this study suggest a protective effect of memantine on memory, preference for social novelty, and depression-like behavior in mice following surgery. However, the study offers a coarse behavioral assay in mice, and further research is recommended to address the mechanisms by which memantine impacts POCD, and the feasibility of using it in humans.