Mytilus galloprovincialis collected along the Central Adriatic Coast  at 25 different sampling sites  located in seven areas. The experimental data, coupled with chemometric approaches , were used to create a statistical model able to discriminate samples as a function of their production site. The LDA model is suitable for achieving a correct assignment of >90% of individuals sampled to their respective harvesting locations and for being applied to counteract fraud.The international seafood trade is based on food safety, quality, sustainability, and traceability. Mussels are bio-accumulative sessile organisms that need regular control to guarantee their safe consumption. However, no well-established and validated methods exist to trace mussel origin, even if several attempts have been made over the years. Recently, an inorganic multi-elemental fingerprint coupled to multivariate statistics has increasingly been applied in food quality control. The mussel shell can be an excellent reservoir of foreign inorganic chemical species, allowing recording long-term environmental changes. The present work investigates the multi-elemental composition of mussel shells, including Al, Cu, Cr, Zn, Mn, Cd, Co, U, Ba, Ni, Pb, Mg, Sr, and Ca, determined by inductively-coupled plasma mass-spectrometry in  Fish products are a valuable source of nutrients for humans. Global consumption of seafood increased steadily from 1961 to 2017, at an annual rate of 3.1%, and aquaculture became a fundamental source of fish production, contributing up to 46% of the total fish market. Among marine animal organisms, bivalve mollusks represent an inexpensive source of protein, characterized by high biological value, and also providing essential minerals, trace metals, and vitamins [The international seafood trade requires food safety, quality, sustainability, and traceability standards. As a result of mussel’s bio-accumulative ability, regular control is necessary to guarantee their safe consumption  in complMussel bioaccumulation of metals, persistent organic contaminants, and biological contaminants is also a very useful marker of environmental pollution, reflecting, in some cases, the impact of anthropogenic activities on ecosystems ,9,10,11.Both the shells and tissues accumulate inorganic contaminants and may be used as indicators; however, several studies have stated that only the shells provide a historical memory of metal content throughout the organism lifetime, which is also preserved after the organism’s death ,102+ and be incorporated in the crystals. Almost any metal present in the environment can be taken up, actively metabolized by the organism, and incorporated into the carbonaceous structure [The mussel shell is a complex organic/inorganic system consisting of two calcified valves coated by an outer organic layer, the periostracum, which supports the initial nucleation for the calcareous shell growth. The shell accumulation of trace metals starts from the cells of a mussel region, the mantle, which secretes the periostracum. A layer of calcium carbonate is laid down on it. As the mollusk grows, the epithelial cells in the inner cavity of the mantle gather calcium and bicarbonate ions from the water, enabling shell building. Other metals, like Mg, Mn, Zn, Pb, and Cu, can substitute the Catructure ,12.The composition of mussel tissues depends not only on the geographical origin, but also on weight, dimensions, and spatial-environmental distribution ,14,15.Several studies have shown that shells and soft tissues have different levels of inorganic element bioaccumulation because, although the shell may incorporate much fewer metals than the soft tissues, their metabolization is lower, enabling element sequestration . The sheGiven all the above statements, the present study aimed to exploit the mussel’s inorganic composition to trace their geographic origin. Therefore, mussel contaminant monitoring, paired with their inorganic fingerprints, may help in the safety tracing of mussels.Mytilus galloprovincialis collected in the Adriatic Sea.An analytical method enabling the analysis of a large set of inorganic elements in mussel shells was optimized by inductively-coupled plasma mass-spectrometry (ICP-MS), and the most relevant elements to be used for a possible geographical origin identification were selected. The elements were analyzed in several mussel shells, and the obtained results were processed by chemometric tools like principal component analysis (PCA) and linear discriminant analysis (LDA). The goal was to create a statistical model able to identify the sample’s geographic origin by means of the inorganic and trace element composition of their shells. To the best of our knowledge, this is the first work dealing with the geographic traceability of Many papers have analyzed trace elements in the soft tissue and byssus of mussels to define temporal and spatial variation ,20,21. RMussels shell pre-treatment and acid digestion needed to be optimized to obtain mineralized solutions free from interferences.2O2 (30%) soaking. After 2, 4, and 6 days, not all the organic material and periostracum seemed to be exhaustively removed from the shell surfaces. Only after 10 days were the valves perfectly clean and easily crunchable, yielding a fine homogenous powder.Experiments were performed in triplicate to optimize the best pre-treatment time  by H2 salt). Other acidic mineralization mixtures were tested without satisfactory results, especially regarding calibration linearity and repeatability. The best conditions were those obtained following EPA Method 3051A [Microwave sample digestion experiments were performed to obtain a clear mineralized solution. Initially, shell samples were digested using nitric and hydrofluoric acid , but a od 3051A .2 > 0.990) and low response factor variability (accepted ±20%). Good trueness and precision were reached in reference material analysis : 3–24%, only Al = 30%). With the optimized conditions, the presented method showed good linearity (determination coefficients RInput data are organized in datasets (matrices), whose rows correspond to sample replicates (objects), while the columns correspond to the measured variables. Each column corresponds to a metal concentration . The correlations and anti-correlations between Zn and U (0.60), Ca and Co (−0.60), Mn and Al (0.57), Ca and Ni (0.56), Co and Mg (0.52), Cd and U (0.51), and Cd and Ba (0.50) were significant but less strong.A PCA model was computed for the matrix containing data from farmed mussel shells (153 rows × 14 columns). The first two PCs explain 36.9% of the total variance. The influence of the different parameters can be evaluated from both the scores plot and the loadings plot. Except for three objects (three replicates of the same samples) in sampling sites “T” and “M”, both characterized by high levels of Sr and Ba, The factors affecting the grouping are unknown and could be related to the different water depths, or the different locations of the production plants with regards to the coast, including the relative position with respect to a river mouth, which may strongly influence the inorganic element water loading.Another PCA model was created for the dataset containing mussels from natural banks (63 objects × 14 variables).For the natural bank shells, In this case, samples from the same production site are not always well grouped. This is particularly true for the samples coming from the Pesaro area (red samples in A classification method (LDA) was applied to the data in order to further highlight the eventual clusters that emerged from the PCA.The first analysis was applied to all samples (dataset with 216 objects × 14 variables), and considering the mussel origin  as an a priori category. The LDA model showed an encouraging NER of 93.5%, with only three farmed objects misclassified as natural and 11 natural objects misclassified as farmed (5 from sampling point “V” and 6 from “Y”). The LDA model gave a 98.0% correct assignment for farmed, and 82.5% for natural growing mussels. Next, data were divided into “farmed” and “natural” samples, in order to check, by LDA, if a stronger discrimination than that observed by PCA was present between sampling sites.A LDA model was calculated for the farmed samples (dataset with 153 objects × 14 variables). The sampling sites were considered the a priori category. The classification method applied to this dataset gave an overall NER of 94.1%: only nine samples out of 153 were misclassified, assigning them to different sampling sites. As shown in the discriminant plot in The values of sensitivity and specificity , which wA LDA model was created from the dataset of natural bank mussel shells (dataset with 63 objects × 14 variables). Similarly, in this case, the a priori category was given to the sampling sites.A further LDA model was computed on the entire dataset, with both farmed and natural samples (dataset with 216 objects × 14 variables). The aim was to evaluate if the sampling sites could be discriminated, regardless of their production method . Thus, in this case, the a priori categories were also the sampling sites. The LDA NER was again very good: 93.5%. Only 14 objects out of 216 were misclassified. Moreover, only three objects from the natural bank samples were misclassified into farmed shells, and only one object from the farmed shells was misclassified as a natural bank samples. The sensitivity and specificity  for all As described above, the implemented statistical model enabled a good classification performance, with a high percentage of correct assignment and efficient discrimination of farmed samples from wild ones. Mussels from natural banks, indeed, grow in an environment significantly different from farmed ones, thus also strongly influencing the aspect and composition of shells. First, the lifetime scale of natural mussels is usually longer and less standardized than farmed mussels, enabling the shells to grow thicker and more robust. Moreover, mussels from natural banks, being closer to the coast and having a larger nutrient availability, share their growing habitat with several other organisms, which also colonize their shells, unavoidably influencing their composition.In Mytilus galloprovincialis species were harvested from 25 sites  along the Marche region coast , in the Adriatic Sea, in the framework of a regional surveillance plan. Natural banks are cliffs where mussels grow naturally, while farms are off-shore breeding sites, located roughly two nautical miles away from the coast. Mussels samples belonging to the 3, 65%, Suprapur®), Hydrogen Peroxide , Hydrofluoric acid , and Hydrochloric acid .The reagents used in sample digestion were Nitric acid .Rhodium (Rh) and Lutetium (Lu) pure standard at 1000 μg mL−1; Perkin Elmer). Quality control spiked samples were prepared using Multi-Element Calibration Standard 3 , a solution of Ca, Mg , and mono-element stock solutions for Al , Ni, Mn, Zn . All solutions were prepared in 5%vv/ HNO3.Matrix match calibration curves were prepared using Multi-Element Calibration Standard 3 .The rinse solution was 5%Certified reference material was included in the analytical batches in order to assess the precision and trueness (accuracy) of the method: Dolt 5, fish liver . 2O2 30% solution for 10 days [3 67–69% and 1 mL of HCl 37% in a high-pressure microwave system (Milestone-Ethos1-HPR1000), using a temperature/pressure programming procedure [The mussel samples were mechanically opened, the soft tissues removed, and the shells rinsed with water. To analyze a sufficiently homogeneous and representative quantity of sample, 4 valves were submitted to pre-treatment by immersion in H 10 days . The drirocedure .The thus obtained digested solution was diluted with ultrapure water, and adjusted to a final volume corresponding to 50 ± 1.0 g in polypropylene vessels. Further dilutions were performed according to the elements to be analyzed. The analyses of Al, Ba, Cd, Ca, Co, Cr, Cu, Mg, Mn, Ni, Pb, Zn, Sr, and U were performed by ICP-MS, on an ELAN DRC II (Perkin Elmer) equipped with an ASX-520 autosampler (Perkin Elmer). The sample was introduced by a peristaltic pump, a Meinhard quartz concentric nebulizer, and a cyclonic quartz spray chamber. The internal standard solution was automatically delivered through a mixing block. Argon (purity 99.999%) was used as the plasma torch, nebulizer, and auxiliary gas, while ammonia (purity 99.99%) was the reaction gas in DRC mode. The ICP-MS parameters were : −1 to 500 µg L−1, choosing for each element the most appropriate range.The calibration curves used for quantification were matrix-matched from 0.1 µg L−1 element/counts sec−1 internal standard) and the respective concentrations, and using the least-squares algorithm. Each calibration curve was created on at least 3 points other than the matrix sample. The best fit curve was obtained by interpolating the response factors , trueness, and quantification limits (LOQ). The last figure of merit was calculated based on signal-to-noise ratio, using the criterion 6σ . The expThe laboratory background contamination level was monitored by analyzing reagent blank samples; accuracy was assessed by batch certified reference material (CRM Dolt5) and spiked sample analysis.The shell matrix was shown to be highly inhomogeneous, therefore batch-to-batch repeatability checking was necessary, and all the samples were analyzed in triplicate in order to have robust results.Pearson correlation matrix was calculated to evaluate correlation or anti-correlation between variables . A direct correlation takes place when the correlation index is positive, otherwise, the variables are anti-correlated . When thPrincipal components analysis is a well-known chemometric technique used for the exploratory analysis of a data structure . It consLinear discriminant analysis (LDA) is a classification approach based on the maximization of the ratio of the between-class variance to the within-class variance in the dataset, thereby pursuing the maximal discrimination between known sample classes . It assuG × G, where G is the number of a priori classes. The diagonal elements ggn represent the correctly classified objects, while the off-diagonal elements represent the objects erroneously classified.Several parameters can be used for the quality estimation of the classification models . All indn is the total number of objects. It is also called accuracy, or classification rate.The non-error rate (NER) is a measure of the quality of a classification model: it represents the percentage of correctly assigned objects. The NER is defined as follows (Equation (1)):gSn) describes the model’s ability to recognize objects belonging to the g-th class and is defined as (Equation (2)):gn is the number of objects belonging a priori to the g-th class. The sensitivity (gSp) is the ability of the g-th class to reject the objects of all other classes and is defined as (Equation (3)):g’n is the total number of objects assigned to the g-th class [The specificity  and 1 (perfect class discrimination) .Person’s correlation matrices and the PCA were calculated by the software CAT , based oA method enabling the simultaneous ICP-MS analysis of 14 elements  in mussel shells after microwave digestion was developed and optimized. The method was applied to mussel shells collected in 25 sampling sites located along the central Adriatic Sea coast, and the obtained results were processed by chemometric tools, in order to discriminate mussels as a function of their production method and site.Among the here applied statistical methods, LDA models were able to achieve a high percentage (>90%) of correct assignments of the samples to a specific sampling point. Therefore, this model could be applied as a useful tool to ensure mussel traceability, counteract fraud, and guarantee product quality.Mytilus galloprovincialis have discriminating power concerning the production method and sampling site. In particular, LDA showed a potentially interesting discriminating power for Co, Ni, Zn, and Sr, due to the high LDA loadings shown in all the computed models. However, to obtain a robust multivariate screening method, further work is needed: the dataset will be implemented with a higher number of samples for each site and for each sampling period, whose eventual influence on results has not yet been explored.Moreover, this work indicates that the contents of Al, Cu, Cr, Zn, Mn, Cd, Co, U, Ba, Ni, Pb, Mg, Sr, and Ca in the shells of


Background: Oxidative stress (OS) plays an important role in the progression of many aging-related diseases. Exercises can delay this kind of progress, but aerobic exercise is the most commonly used type of training among older adults; therefore, its influence needs to be further verified.Methods: A literature search was conducted in eight electronic databases, including Cochrane, EMBASE, PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP), Wanfang Date, and SinoMed from their inception to April 2020. Methodological quality was assessed using Cochrane RoB tool v2.0 for individual studies, and RevMan 5.3 software was used to perform the meta-analysis.Results: The meta-analysis included 20 studies, involving 1,170 older adults. The results showed that regular aerobic exercise could reduce blood oxidant markers, including malondialdehyde  and lipid peroxide , and increase the levels of antioxidant factors, such as nitric oxide , superoxide dismutase , and total antioxidant capacity , with clear statistical significance. It may also improve the levels of other OS markers, such as 8-OHdG, 8-isoPGF2, VE, and reduced glutathione/oxidized glutathione (GSH/GSSG).Conclusion: Regular aerobic exercise may have a positive effect on the OS levels of older adults by reducing some oxidant markers and increasing antioxidant marker levels. Aging is a dynamic, degenerative, biological, and time-dependent process. It is characterized by the gradual accumulation of damage to cells, progressive functional decline, and increased susceptibility to disease . Therefo2−), hydroxyl radical (OH), hydrogen peroxide (H2O2), and peroxynitrite  to inactivate it; the OONO− formed by the reaction can oxidize NO synthase and make it unstable. And this unstable NO synthase will further increase the production of peroxide , China Science and Technology Journal Database (VIP), Wanfang Date, and SinoMed, from their inception to April 2020, without restrictions on language or publication type. The original search covered a combination of relevant keywords relating to aerobic exercise , OS , and older adults . In addition, the references of all articles included in the study were checked to identify other relevant articles. The complete search strategy can be found in the Studies were included if: (1) the design of the study was a randomized controlled trial; (2) participants were older adults aged 60years old or older, no gender, or race restrictions; (3) the intervention in the experimental group could be any form of aerobic exercise  or aerobic exercise combined with nonexercise intervention measures ; however, aerobic exercise should occur at least three times a week, at least 20min each time and for no less than 4weeks; (4) the control group had either no specific exercise intervention , or the same nonexercise intervention as the experimental group; and (5) outcome measures included one or more OS markers measured in serum or plasma.All the searched records were imported into the reference management software (EndNote X9.3.2). After eliminating duplicated records using software, two researchers (YY and PTQ) independently checked and selected the eligible studies by reading the titles, abstracts, or full text based on the predefined eligible criteria. Any disagreements were resolved by discussion with the third reviewer (GHZ). Data in the eligible studies were extracted by one reviewer (YY) using the predesigned form and it was checked for accuracy by another reviewer (HYL). The extracted information included participants’ characteristics, study design, sample size, methodological information of study quality, aerobic exercise parameters , controls, outcomes and their measurement method, and adverse events.Two reviewers (YY and MYW) independently assessed the risk of bias of the included studies using the revised Cochrane Collaboration’s risk of bias tool (RoB V.2.0) for RCTs . The fiv2 value. With the Chi-square test, p<0.05 was considered to be significant. If there was no significant heterogeneity among studies, the pooled effect was calculated using the fixed-effect model. Otherwise, the random-effects model was applied , we calculated the combined means and SD according to the Cochrane guidelines. The size of the effect from baseline to postintervention for each OS marker was expressed as the mean difference (MD) and its 95% CI if the measurement method was the same; if not, the standardized MD and its 95% CI were used . The het applied . The reaAccording to the search strategy, 16,833 citations were retrieved from the eight databases. After removing duplicates, two reviewers (YY and LHY) screened titles and abstracts to exclude unrelated records. Then, 181 full-text articles were assessed for eligibility according to the inclusion criteria. Finally, 20 studies involving 1,170 older adults were included in the meta-analysis . The detThere were 20 randomized controlled trials involving 1,170 older adults, including 546 male subjects and 624 female subjects, with an average age of 62.3–72.4years old. The types of aerobic exercise used in the experimental group were diverse, most of which were walking  or combip<0.001, I2 =86%, seven studies, 398 samples). Subgroup analysis based on exercise type showed that both body–mind exercise and other aerobic exercise types could significantly decrease the MDA levels. Furthermore, no particular study prominently affected the overall effects by the sensitivity analysis ; no significant differences were found in the levels of 3-NT or Ox-LDL markers between the two groups .Seven studies investigated the effect of aerobic exercise on MDA. Meta-analysis showed that aerobic exercise led to a significant decrease compared with the controls, with high heterogeneity across studies , with obvious heterogeneity (I2 =83%). Subgroup analysis based on different exercise types (mind–body exercise or other aerobic exercise types) or intervention methods  also showed a significant improvement in the NO level, except for the subgroup of aerobic exercise interventions alone . However, the sensitivity analysis for the subgroup of aerobic exercise interventions alone showed a significant improvement when the study by p=0.001, I2 =72%, seven studies, 448 samples; SMD-TAC=1.22, 95% CI 0.45–1.98, p=0.002, I2 =74%, four studies, 164 samples). Subgroup analysis of SOD based on different exercise types (the mind–body exercise or other aerobic exercise types) showed that mind–body exercise, but other aerobic exercise types had a significant effect on improving the SOD levels  or glutathione (GSH) levels  between groups. In addition, one study reported a significant increase in vitamin E levels , but another study found a significant decrease in GSH/GSSG levels  in the aerobic exercise intervention group compared with the control group.Seven studies investigated NO levels in plasma or serum. The pooled results showed a significant increase in the aerobic exercise group compared with the control group , and most of them ranged from 40 to 80% HRmax; thus, they belong to low-moderate intensity with VO2max less than 75% , a transcription factor that is modified by the redox state of the cell, participates in cellular responses to OS status and ROS-producing agents , and inhradicals . Numerouradicals . A cliniradicals . TAC mayoxidants . Severaloxidants . In our To the best of our knowledge, this is the first systematic review and meta-analysis to investigate the impact of regular aerobic exercise on OS levels in older adults. Most previous studies were narrative reviews and focused on exercise involving a very broad exercise type  and were also a potential factor for the difference in results. Third, the duration, frequency, and type of aerobic exercise in the experimental group varied widely, which made it difficult to draw accurate and recommendable conclusions. Fourth, six indicators, including Ox-LDL, 8-isoPGF2, 3-NT, VE, GSH/GSSG, and GSH, were measured in only one study, so the pooled effect could not be analyzed. Finally, there may have been potential reporting bias in this systematic review, because its protocol has not been registered previously. In addition, the sample size of nine included studies was less than 50, which may have caused a type II error ; additioThe current systematic review and meta-analysis reveal that regular aerobic exercise may be effective for reducing oxidant marker levels  or increasing antioxidant marker levels . However, considering the significant heterogeneity among studies, these findings should be interpreted with caution.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LC, GZ, and JT conceived and designed the study. YY and PQ participated in the literature search. YY and HL contributed to the analyses. YY and MW completed the methodological assessment. YY and GZ analyzed the data and wrote the manuscript. RX and JH were invited to comment and contribute changes. All authors contributed to the article and approved the submitted version.The work was supported by National Natural Science Foundation of China (Grant No. 82074510).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Reminiscence therapy (RT) is a form of cognitive stimulation therapy that incorporates discussion of past activities, events, and experiences to stimulate individual memories; it has had some success in treating persons with neurocognitive disorders. This research aims to evaluate the ability of individual RT, using a simple reminiscence format, to improve the overall cognitive function, memory, executive functions, emotional status, and quality of life in older adults with neurocognitive disorders who received social care and support services. A multicenter randomized controlled trial was completed in the Azores archipelago  using repeated measures . The intervention group underwent individual RT sessions, twice weekly for 13 weeks, while the control group completed regular activities administered as part of their program. Results did not reveal any significant differences between the intervention and control groups. While results did not reveal significant effects, a number of historical and contextual factors are considered as possible explanations for the lack of effects—namely, data collection occurring during the COVID-19 global pandemic, participant cohort effects, and therapist heterogeneity. Diagnostic Statistical Manual of Mental Disorders, 5th edition (DSM-5) as a cognitive decline relative to a previous level of functioning [Major neurocognitive disorder (mNCD), characterized by the ctioning , is one ctioning . The absOne form of non-pharmacological therapy, cognitive stimulation, seeks to provide an enriching and engaging environment for participants. In NCD samples, this therapy has demonstrated improvements in cognitive functioning, social functioning, and quality of life (QoL) ,4. CogniReminiscence therapy (RT) is a form of cognitive stimulation that incorporates discussion of past activities, events, and experiences, usually with the help of cues  that serve to stimulate individual memories. This method has had some success in older adults with mNCDs (mixed dementia samples) given relatively preserved remote autobiographical memories in this group ,7. The gA Cochrane review described positive, albeit small, effects of RT on cognition, QoL, communication, and possibly on the mood of persons with dementia . DespiteA standardized individual RT protocol for Portuguese-speaking patients with NCD was created, piloted, and implemented, which is detailed elsewhere . ExploraThe current study evaluated the effectiveness of individual RT (iRT) on cognitive functioning , emotional state (depression and anxiety), and QoL in older adults with mNCD. Building on the previous studies using the current iRT protocol, the current study expands in multiple ways. First, participants were from a day program in the Azores Autonomous Region, an archipelago of Portugal that is one of the four Portuguese NUTS II regions that are considered less developed . We alsoThis study analyzed a clinical trial of iRT for people with NCD , which was designed as a multicenter, single-blind, randomized, parallel two-arm , controlled trial. Participants in the intervention group received two 50 min weekly sessions of iRT for 13 weeks in addition to their regular daily activities. Participants in the control group underwent only their regular activities. Participants were assessed at baseline (T0), after the iRT intervention (T1), and at a 3-month follow-up (T2). Participants understood that participation in the study was voluntary.Recruitment took place from 15 to 19 February 2021. Recruitment included contacting social care institutions in the Azores, through the supervising government entity, to participate in an RCT on iRT for people with NCD; overall, 14 care institutions showed interest in the study and contacted the research team, and 12 institutions agreed to collaborate. The institutions were then asked to select participants they believed could participate in the study based on the inclusion criteria, resulting in 170 participants completing the eligibility assessment. After baseline assessment, 122 participants were selected from 12 institutions: 62 (50.8%) allocated to the iRT group and 60 (49.2%) to the control group.Inclusion criteria included a diagnosis of neurocognitive disorder (major or minor) from their general practitioner based on DSM-5 criteria; reviewed and signed consent form; intact language expression and comprehension; access to autobiographical information about the participants through family members or caregivers (via the socio-family questionnaire as part of the protocol); aged 65 years or older; being a native Portuguese speaker; regularly attending an institution that provides social care and support services for older adults.Exclusion criteria included an acute or severe illness that prevented participation in the intervention sessions; severe sensory and physical limitations; severe disconnection from the environment and minimal attention span; the presence of severe neuropsychiatric symptoms or uncontrolled delirium that prevented participation in the sessions; traumatic life history or experienced adverse events that discouraged participation; history of adverse reactions during iRT sessions or similar activities; severe or total functional dependence .® RandList  by one of the study principal investigators blinded to baseline scores and demographics of the participants. The overall number of participants in each institution ranged from two to eight persons. Groups allocation was unknown to participants, therapists, or institution staff until the intervention started. Evaluators remained blinded to participant allocation until endpoint assessments were completed. Enrollment of participants was performed by the researchers responsible for communicating with the institutions who assessed eligibility. The therapists at each institution administered the intervention.Participants who met the inclusion criteria were enrolled, and baseline assessments were completed. Participants were then randomly allocated to either the control group or the experimental group. Due to the multicenter nature of the study, each institution had two groups, the intervention and control groups, with a 1:1 ratio. A non-stratified permuted block randomization process (with variable block size) was carried out using the software DatInfAs noted, the iRT protocol used in this study has been described elsewhere, including specific information on activities and materials for each session . It inclAll iRT sessions were conducted by 14 therapists  who completed a 6 h training on the protocol. Principles of the therapy were discussed by two of the principal investigators, and the materials, objectives, agenda, and activities of each session were provided and reviewed.The sessions were held at each institution. Each therapist scheduled the individual sessions, preferably in a room with minimal distractions. Each therapist always treated the same patients . The intervention lasted 13 weeks, from 1 March 2021 to 28 May 2021. The follow-up period ended on 20 August 2021.The participants in the control group did not receive the iRT intervention but participated in their usual activities provided by the institution. This treatment, although varied according to the staffing available in each institution, included social interaction activities, cognitive stimulation activities, stimulation of personal skills, and administration of any prescribed dementia medication.The protocol included several tools and was administered to all participants (intervention and control groups) by trained evaluators who were blinded to the participant group. Data were collected at baseline (T0), 15 weeks post-baseline (endpoint assessment T1), and 3 months post-intervention completion (T2). The outcome measures addressed the global cognitive function, other specific cognitive functions (memory and executive function), mood (depression and anxiety), functional abilities (only used to assess for eligibility), and QoL.The Mini-Mental State Examination  assessed global cognitive function. Scores range from 0 to 30, with higher scores indicating better cognitive functioning ,18,19.The Memory Alteration Test  assessed memory function. It is an easy and quick instrument that assesses five memory domains: temporal orientation, encoding, semantic memory, free recall, and cued recall. Total scores range from 0 to 50, with higher scores indicating better memory ,21.The Frontal Assessment Battery  assessed executive function in several subtests: conceptualization, mental flexibility, motor programming, sensitivity to interference, inhibitory control, and environmental autonomy. Scores range from 0 to 18, with higher scores indicating better executive functioning ,23.The Geriatric Depression Scale-15  measured depressive symptoms. It is considered a reliable tool to screen depressive symptoms in older adults, in a dichotomous format (yes/no answers). Scores range from 0 to 15, with higher scores indicating more severe depressive symptoms ,25,26.The Geriatric Anxiety Inventory (GAI)  measured anxiety symptoms. It is a self-report measure considered a reliable tool to screen for symptoms of anxiety in older adults. Scores range from 0 to 20 with higher scores indicating more severe anxiety symptoms ,28.The Quality of Life in Alzheimer’s Disease scale  evaluated QoL. This 13-item scale assesses the QoL in people diagnosed with dementia, gathering information from the patient about the following domains: perceived health, mood, physical condition, interpersonal relationships, hobbies, decision-making skills, and life as a whole. Scores range from 13 to 52, with higher scores indicating better QoL ,30.t-tests for continuous variables were performed to determine whether the groups were homogenous prior to treatment. No imputation of missed data was made; thus, only data from participants who completed the follow-up assessment were analyzed.Chi-square tests for categorical variables and p < 0.05 for all analyses. Statistical analysis was performed using IBM SPSS Statistics .The effects of iRT on outcomes  were analyzed using 2 × 3 repeated-measures mixed ANOVAs, with group assignment as a between-subjects factor  and time as a within-subjects factor . The main effects of interest were the group × time interactions. The Greenhouse–Geisser correction was used when the sphericity test was significant. The level of significance was set at Of the 62 participants in the iRT group, 40 (64.5%) completed the three assessments. The intervention could not be completed for 20 participants (32.3%) who were not assessed at T1; two other participants (3.2%) failed to complete follow-up and were not assessed at T2 see . These pOf the 60 participants in the control group, 4 (6.7%) did not complete the endpoint assessment and 2 others (3.3%) could not be assessed at follow-up, resulting in a total of 54 participants (90%) completing the three assessments. The reasons for withdrawal included two leaving the institution where the study was conducted and four being hospitalized.For those participants who completed the follow-up assessment, no significant differences were found between the intervention and control groups regarding demographic variables, clinical condition, or baseline outcome mean scores.F = 2.158, p = 0.126, ηp2 = 0.023.The 2 × 2 ANOVA  did not F = 1.398, p = 0.250, ηp2 = 0.015.ANOVA for FAB scores  did not F = 0.361, p = 0.673, ηp2 = 0.004.The ANOVA for MAT  did not F = 0.013, p = 0.983, ηp2 = 0.000, since both iRT and control groups significantly improved their scores at T1.ANOVA for GDS-15  did not F = 0.067, p = 0.936, ηp2 = 0.001, since both groups reduced their scores through the trial.The ANOVA for GAI  did not F = 0.044, p = 0.957, ηp2 = 0.000 was found in the ANOVA for QoL-AD  of them. Only in 164 (13.4%) sessions did participants appear passive, and only in 18 (1.5%) did participants not cooperate, showing drowsiness or a very low level of attention and concentration that prevented an adequate performance in the proposed activities.This study presents the results of an RCT on the immediate and 3-month follow-up effects of a 13-week iRT intervention for people with mNCDs. The results did not show a significant effect of the iRT intervention on global cognition (assessed through MMSE), though the iRT group improved their scores upon completion of the intervention. No significant effects for the intervention were found on any of the other outcomes, including memory, executive functions, mood (anxiety and depressive symptoms), or QoL. This is an unexpected result, as the current iRT protocol has shown positive effects on global cognition, memory, and QoL in other Portuguese-speaking populations with NCD ,12.Although there was moderate adherence to the intervention  and a high degree of participation in the iRT sessions (participants collaborated in 85.1% of sessions), the number of dropouts was high (32.2%). Some studies that found no significant effects of iRT had a dropout rate of over 40% and low adherence .There are a number of other factors that could explain our lack of significant treatment effects. First, the intervention and data collection occurred during the COVID-19 global pandemic, which is likely to have affected mood and cognitive functioning given associated health concerns and isolation, particularly on an island with travel mostly occurring by air (and imposed restrictions). During the period of the COVID-19 global pandemic, in the initial phase, participants saw confinement and isolation measures, while in the final phase, restrictions were somewhat eased, including restarting family visits. Finally, cohort effects might have played a role. The current sample experienced a political revolution in the 1970s in which a military coup overthrew an authoritarian regime, and the Azores islands were granted autonomy by the Portuguese government; thus, cultural and personal past events may not have been experienced similarly in the current sample relative to the past participants who had positive effects from the iRT, despite making some prior modifications to the content of the intervention (photos) to mitigate past experience differences. Lastly, as previously stated, the effectiveness of reminiscence therapy has been mixed [The study had some limitations that must be considered in the interpretation of its results and implications. The medium size of the sample and relatively short intervention duration may have limited our capacity to detect cognitive or behavioral changes that could have emerged with a longer intervention. Additionally, we had limited information on the participants’ medications or other treatments received. Regarding diagnosis, the lack of confirmation through biomarkers makes it possible that those with other amnestic syndromes (e.g., Limbic-predominant age-related TDP-43 encephalopathy , were inOverall, the current iRT intervention findings provide important contributions to the literature on this topic. While our results revealed no significant effects, it adds to our understanding and exploration of the current iRT protocol  that wil


Considering the high incidence and mortality of breast cancer, a lot of trials evaluating new drugs for the treatment of this disease are currently ongoing. However, few drugs show a statistically and clinically significant improvement in the outcomes leading to the Competent Authority approval. In this review we analyzed the European Medicines Agency breast cancer drug indications issued between January 2015 and June 2021 and we evaluated the clinical trials results leading to the approval and their update. Breast cancer represents the first cause of cancer worldwide and the leading cause of cancer mortality for women. Therefore, new therapies are needed to improve the prognosis of women diagnosed with this disease. In this review, we summarize the new drug indications for the treatment of breast cancer approved by European Medicines Agency between January 2015 and June 2021. In particular, we analyzed the clinical trials results leading to approvals and their update (when available), according to setting  and clinical features . The aim of this paper is to describe the clinical benefit obtained with the new indications.  Breast cancer represents the most frequent cancer and the leading cause of cancer-related mortality in females . In thisConventionally, this disease includes three major subtypes: hormone receptor positive, which represents about 70% of cases; human epidermal growth factor receptor 2 (HER2) positive (15–20%); and triple negative (15%). More than 90% of cases are not metastatic at diagnosis and for stage I, the 5-year breast cancer-specific survival is about 99% for hormone receptor-positive breast cancer, ≥94% in HER2-positive disease, and ≥85% in triple-negative patients. Instead, for metastatic disease, median overall survival is 4–5 years for the hormone receptor-positive subgroup, 5 years for HER2-positive disease, where the prognosis is remarkably improved with the specific therapies, and 10–13 months for triple negative  population and PD-L1–positive subgroup) and OS . In the study, the experimental arm showed an advantage in PFS and a non-statistically significant improvement in OS in the ITT population  . The EMAIn BRCA 1/2-mutated cancers, two PARP inhibitors obtained approval: olaparib and talazoparib. Both are approved in monotherapy for the treatment of BRCA-mutated HER2-negative locally advanced or metastatic breast cancer patients previously treated with an anthracycline and a taxane in the (neo)adjuvant or metastatic setting and endocrine therapy if hormone receptor positive.p < 0.001) with a good toxicity profile; in particular, grade 3 or higher adverse events were reported in 36.6% and 50.5% of patients in the experimental arm and standard chemotherapy, respectively, and the most common were hematologic events. The rate of treatment discontinuation for toxic effects was 4.9 and 7.7%, respectively. The OS had not yet reached 50% of the events, but it did not differ significantly  [p = 0.513) [Olaparib was tested in the OlympiAD trial, where 302 previously treated patients  with germline BRCA 1/2 mutation were randomized in a 2:1 ratio to receive the PARP inhibitor or the investigators’ choice chemotherapy with capecitabin, eribulin, or vinorelbine. Olaparib showed an advantage of 2.8 months in median PFS  .p < 0.001), and OS, one of the secondary endpoints, was 22.3 and 19.5 months . The incidence of grade 3–4 hematologic adverse events was higher in the experimental arm (55% vs. 38%), while grade 3 non-hematologic adverse events occurred in 32% and 38% of patients, respectively [p = 0.17) [Indeed, talazoparib was evaluated in the phase III EMBRACA trial in patients with germline BRCA 1/2 mutation, previously treated with no more than three lines of chemotherapies . In total, 431 patients were randomized in a 2:1 ratio to receive talazoparib or the physicians’ choice chemotherapy . Median PFS, which was the primary endpoint, was 8.6 versus 5.6 months, respectively  .Breast cancer, along with lung cancer and melanoma, is one of the principal tumors for which new drugs have been approved by EMA in the last years.Our analysis showed that only one approval in the locally advanced setting and one in the metastatic disease were based on phase 2 trials and none on phase 1 trials.The majority of approvals were in metastatic disease (76%); in this setting, more than 60% were issued in hormone receptor-positive/HER2-negative patients and of them, 80% concerned a CDK4/6 inhibitor. Instead, in localized and locally advanced tumors, all approvals concerned HER2-positive disease.An interesting consideration is that, after more than 20 years of trastuzumab approval, new drugs have entered the breast cancer treatment landscape based on biomarker expression. In particular, for the first time in triple-negative disease, two biomarkers are considered in clinical practice: PD-L1 and BRCA. In luminal tumors, after over 50 years, new biological molecules have been approved other than endocrine therapy and a new biomarker, PIK3CA, is being evaluated for specific treatment. Finally, for HER2-positive tumors, HER2 expression remained the unquestioned target, with two new drugs, the tyrosine kinase inhibitor tucatinib and the antibody drug conjugate trastuzumab deruxtecan, that entered the therapeutic scenario of this disease .Regarding primary endpoint, among 20 clinical trials leading to the 21 approvals, only one considered OS as a primary endpoint ,35. In pConcerning clinical efficacy, almost all trials demonstrated an important improvement in the outcome. In the adjuvant setting, the experimental arm showed a reduction of 19% and 33% for 2-year IDFS in the APHINITY and ExteNET trials ,19,20,21In metastatic disease, the CDK4/6 inhibitors associated with endocrine therapy radically changed clinical practice due to a remarkable improvement in terms of PFS compared to hormone therapy alone of 9–13 months in first line ,16,24,25Among 14 trials leading to drug approval in metastatic breast cancer, only three reported OS results at the approval time. In particular, tucatinib showed a significant advantage of 4.5 months , atezoliConsidering the high incidence of breast cancer, medical and commercial interest focused on this disease has led to an investment in research in this pathology. A lot of innovative drugs have been approved for the treatment of breast cancer in the last years, with the majority of cases showing impressive results in terms of PFS and/or OS. In particular, the big novelty of locally advanced breast cancer treatment was the introduction of the HER2 double block, so that clinicians can now be confident in treating an earlier stage of cancer with a pre-operative strategy. In advanced luminal breast cancer, CDK4/6 inhibitors demonstrated a remarkable improvement in PFS and OS, and recently, the biomarkers PIK3CA and BRCA have defined a new strategy of treatment. In advanced HER2-positive tumors, we have new sequences due to the evidence of the significant impact of PFS with new molecules from the third line. In triple-negative breast cancer, the two new biomarkers, BRCA and PD-L1, have led to the use of specific therapeutic strategies.Finally, some drugs have demonstrated little improvement in clinical outcomes, in some cases without an OS advantage. However, other factors, such as quality of life, toxicities, manageability, and costs (direct and indirect), should be considered. Moreover, a regular safety and efficacy monitoring of approved drugs should also be organized to establish the real benefit of the new indication.In conclusion, thanks to these approvals, we are seeing a magnitude of clinical benefits for breast cancer patients. However, a careful selection of those who could really benefit from a treatment through the identification of biomarkers predictive of efficacy should be recommended.


Root zone soil moisture (RZSM) is an essential variable for weather and hydrological prediction models. Satellite-based microwave observations have been frequently utilized for the estimation of surface soil moisture (SSM) at various spatio-temporal resolutions. Moreover, previous studies have shown that satellite-based SSM products, coupled with the soil moisture analytical relationship (SMAR) can estimate RZSM variations. However, satellite-based SSM products are of low-resolution, rendering the application of the above-mentioned approach for local and pointwise applications problematic. This study initially attempted to estimate SSM at a finer resolution (1 km) using a downscaling technique based on a linear equation between AMSR2 SM data (25 km) with three MODIS parameters ; then used the downscaled SSM in the SMAR model to monitor the RZSM for Rafsanjan Plain (RP), Iran. The performance of the proposed method was evaluated by measuring the soil moisture profile at ten stations in RP. The results of this study revealed that the downscaled AMSR2 SM data had a higher accuracy in relation to the ground-based SSM data in terms of MAE (↓0.021), RMSE (↓0.02), and R (↑0.199) metrics. Moreover, the SMAR model was run using three different SSM input data with different spatial resolution: (a) ground-based SSM, (b) conventional AMSR2, and (c) downscaled AMSR2 products. The results showed that while the SMAR model itself was capable of estimating RZSM from the variation of ground-based SSM data, its performance increased when using downscaled SSM data suggesting the potential benefits of proposed method in different hydrological applications.  Spatiotemporal distribution of root zone soil moisture (RZSM) across massive areas of land significantly contributes to lots of meteorological, hydrological, and agricultural applications ,2. MoreoHaving gathered in the field, soil moisture (SM) measurements—typically known as in situ measurements—can be generally retrieved from low- or high-density networks of point measurements. To determine network density, the amount and assembly of tools, project budget, and study area should be taken into account . PointwiRecent technological and theoretical advancements have paved the way for using remote sensing techniques in measuring SM content. Active and passive microwave observations have been widely used to estimate SM e.g., ,10,11) a,11 a10,1Satellite-derived remotely sensed SSM products have their own advantages, including the accessibility of global-scale measurements at a continuous spatiotemporal resolution (STRs). Nevertheless, satellite-based products are currently accessible at low spatial resolutions, rendering them useless for small-scale agricultural applications and hydrologic models ,15. The Microwave sensor-derived SM data are immediately associated with the surface soil layer (0.2 cm to 5 cm) ,26. HoweBy solving a simple water balance equation for arid and semi-arid zones, ref.  developeThe spatiotemporal accessibility and accuracy of SSM and RZSM measurements are significant components to obtain optimal results in a variety of applications. There are also extremely restricted resources accessible to reclaim accurate SM measurements in the Rafsanjan plain (RP). Thus, this study will assess AMSR2 data quality using REC-P55 SM sensors manufactured by Ansari and Hassanpour  and applVIS/NIR, TIR, and microwave are currently the three main data sources for global SSM monitoring. The VIS/NIR and TIR data have higher spatial resolution and can easily be affected by clouds, while microwave data have lower spatial resolution and can provide all-weather data coverage. To overcome the gaps between these sources of data, the synergic multi-band SSM retrievals should be considered.Considerable efforts have been made in the retrieval of two Land Surface Temperature (LST) and SSM using passive microwave observations and auxiliary data. However, SSM and LST are codependent making the retrieval process difficult. Moreover, obtaining accurate auxiliary information such as meteorological parameters and soil texture is difficult due to clear-sky dependency and low spatial resolution, respectively. To solve this problem, it is essential to simultaneously retrieve LST and SSM from only passive microwave data to reduce the number of unknown parameters and make the retrieval independent of auxiliary information.3/m3 between the satellite and measured SSM data has been the criterion for validating the SSM products, however, scientists suggested that distinguished target accuracies should be determined for different combination of surface moisture status, soil texture, and vegetation coverage.The target accuracy of RMSE of 0.04 mIn order to validate remotely sensed SSM data, in situ SSM measurements with a fixed depth of 5 cm are frequently used. However, VIS/NIR and microwave data can reflect SSM with only a few millimeters and centimeters, respectively, mostly depending on SSM content within the soil column and frequencies used for detecting SSM. Therefore, it seems that currently there is no good way to solve the contradiction of sensing depth, however, post-processing steps  can mitigate this problem.The estimation of RZSM from satellite SSM has been increasingly proposed by a number of investigations. The possible approaches for obtaining such data are (a) assimilating microwave SSM data into land surface models; (b) exponential filter and the analytical relationship between RZSM and SSM; and (c) using the P-band SAR with much deeper penetration depth due to its longer wavelength. However, satellite SSM accuracy and soil texture distribution are among the great challenges to obtain RZSM at present.At the end, a summary of the established and emerging soil moisture retrieval methods as well as the scientific challenges seem necessary to understand the milestones in this field of study. According to a latest review, the state-of-the-art topics can be summarized into five items :VIS/NIR2. The study area is located between 55°1′ to 56°28′ longitude and 30°3′ to 31°11′ latitude, where the elevation ranges from 1296 to 2131 m above sea level (ASL). The average annual rainfall and average annual potential evapotranspiration (PET) here are below 100 mm and above 3000 mm, respectively. There is a thermic soil temperature regime and an aridic SM regime here.In this study, the Rafsanjan Plain was taken as the area of study, situated in southeast Iran, Kerman Province , Iran’s The Global Change Observation Mission–Water 1 (GCOM-W1) satellite system was launched on 17 May 2012, to gather geophysical parameters, such as precipitation, sea surface temperature (SST), and SM, and witness variation in water circulation . It carrhttps://hydro1.gesdisc.eosdis.nasa.gov/) (accessed on 18 April 2018). The complete ID of the product is: AMSR2/GCOM-W1 surface soil moisture (LPRM) L3 1 day 25 km × 25 km ascending V001 (LPRM_AMSR2_A_SOILM3)  represents top soil porosity, Lr1 (L) represents top soil depth, In the above equation, I (t) [[–] represents the amount of saturated soil penetrating the lower layer, ce layer . The depce layer ,48,49,502 [[–] soil porosity, By defining equation :(7)s2(tjThe SMAR parameters for different soil textures are shown in Three input sources were envisaged for the SMAR model (three schemes) in order to investigate the effect of spatial resolution of the results: (1) the SMAR model based on ground-based measured SSM, which estimates RZSM values at selected stations, (2) the SMAR model based on AMSR2 25 km SSM, and finally (3) the SMAR model based on downscaled 1 km SSM data using MODIS products.The AMSR2 SSM product at a 25 km spatial resolution was downscaled to 1 km spatial resolution using a simple linear equation containing MODIS Albedo, LST, and NDVI products . The fol2 = 0.61 and the acceptable p-value (<0.05) and F-statistic test . The regression model was obtained with Rp-values are below the significance level of 5%, meaning that the null hypothesis for each coefficient is rejected and all components are significant in the linear equation. The results indicated that the downscaling model offered an excellent fit with the AMSR2 estimates.The AMSR2 25 km SSM and downscaled SSM values were examined by calculating MAE, RMSE, and R for every station. According to Other factors such as changes in vegetation density, land cover, land use, etc., at AMSR2 25 km pixel could also affect the results of MAE, RMSE, and R. Evaluating the land cover map of the study area a showed According to 2) in the SMAR model, the V2 value was obtained 5.8 mm/day regarding optimization based on regional conditions using the genetic algorithm (GA) in MATLAB programing environment, with RMSE function between simulated RZSM and observational values.Three of the four SMAR parameters  were determined based on soil texture map of the study area b, accordsub) based on the SMAR model in the Rafsanjan Plain for 29 August 2018.The results indicated an increased accuracy of the model in estimating RZSM using the downscaled SSM data obtained from the method proposed in this study. In this method, mean R, RMSE, MAE values between all the stations were obtained equal to 0.71, 0.032, 0.032, respectively. Since SSM is considered as the most critical parameter of the SMAR model provided that it could be estimated with high accuracy, it will affect the more accurate estimation of RZSM by the SMAR model, verified by the results obtained. The downscaled SSM-based SMAR model can be utilized to develop a system for receiving downscaled SSM values to create RZSM maps. Hence, the AMSR2-downscaled SSM model was used in the SMAR model based on MODIS parameters, and the RZSM values were obtained at 1 km pixels for different days. This study developed an RZSM estimation model, i.e., SMAR, at 1 km × 1 km pixels in the Rafsanjan Plain. Initially, the AMSR2 25 km SSM was compared with the ground-based SSM values in the Rafsanjan Plain. The overall validation results suggested a low correlation in most stations. In addition to the distance of the station from the midpoint of 25 km pixel, other factors such as changes in vegetation compaction, land-use, topography, rainfall, and soil properties of the study area could affect the correlation between large-scale data and measured field data. It is necessary to collect more SSM data from 25 km pixels to have stronger validation.The coarse-resolution satellite-based SSM data necessitates the downscaling of SSM data for many hydrological and agricultural plans in the study area, contributing to a precise RZSM estimation by the SMAR model on a finer scale.Since fine-resolution MODIS data are available globally and daily, this study considered the downscaled AMSR2 SSM based on MODIS parameters. The results demonstrated a good consistency between the downscaling method that exploits these parameters and the measured data—which were consistent with those of the previous studies ,52—leadiTo obtain RZSM at 1 km pixel in the Rafsanjan Plain, the downscaled SSM model was used based on MODIS parameters in the SMAR model. RZSM obtained from the SMAR model based on downscaled SSM was compared with measurement RZSM data. The results indicated that the model could initially manage to estimate RZSM values from SSM variations, and then increase the accuracy of the model to estimate RZSM using the downscaled data obtained from the proposed method in this study. Mean R, RMSE, MAE values were obtained for all the stations equal to 0.71, 0.032, 0.032, respectively. As the most crucial parameter of the SMAR model, the accurate estimation of SSM will affect the more accurate estimation of RZSM by this model .It is essential to estimate RZSM for different biogeochemical, ecological, and hydrometeorological applications and modeling. This study developed the SMAR model for estimating RZSM at 1 km pixels of the Rafsanjan Plain and subsequently, evaluated its variations. Spatiotemporal continuity of estimated RZSM by this model is one of the advantages of this method. SSM is the most important input parameter in the SMAR model, which will lead to a more accurate estimation of RZSM if precisely determined. Methods based on microwave remote sensing are suitable for retrieving SSM.It is suggested to estimate other effective parameters of the model, e.g., soil moisture loss coefficient and soil texture parameters, more accurately for each pixel in the Rafsanjan Plain, optimize them with measured data, and use them in the SMAR model. It was generally concluded that the SMAR model, in which SSM was downscaled based on MODIS parameters, could accurately estimate and demonstrate the variations of the RZSM of the Rafsanjan Plain.It is also suggested to consider other affecting variables, including rainfall that improves downscaled SSM products. Consider the effect of other VIs, including soil-adjusted vegetation index (SAVI) or simple ratio vegetation index (SRVI) on improved quality of downscaled products, and utilize the best of these indices. Optimization of downscaling approaches using sensitivity analysis (SA) by a network of sensors within the coverage area of a single AMSR2 pixel is suggested to validate the AMSR2 products and downscaled SSM values in the future studies.


Odontogenic keratocyst (OKC) is a development cyst, of odontogenic origin, that differs from other entities by its infiltrating and aggressive biological behavior. Among conservative treatments for large lesions, surgical decompression stands out, with a variable recurrence rate. Aim: To evaluate the histological effects of decompression treatment on OKC, including cell proliferation and apoptosis of epithelial cyst. 21 OKC cases were included. Samples were taken before and after surgical decompression for histological evaluation and immunohistochemical staining of Ki-67, MCM4/7, Bax and Bcl2. Data were analyzed and compared using Student’s t and Wilcoxon tests for related samples, and p values <0,05 were considered statistically significant. p=0,029), loss of parakeratinization of the epithelium  and absence of palisade cell distribution in the basal layer were observed . There were no statistically significant changes in the expression of Ki-67 , MCM4/7 , Bax  or Bcl-2 when compared before and after decompression. After decompression treatment an increase in inflammation of the cystic wall (Surgical decompression generates histological structural changes in OKC both in the epithelium and connective wall, however, these findings do not seem to alter induction of the cell cycle or epithelial apoptosis. Key words:Odontogenic keratocyst, MCM, Bax, Bcl2, Ki-67, apoptosis, decompression.  As described by the World Health Organization , odontogOKC differs from other developmental odontogenic cysts due to its infiltrative and aggressive biological behavior, associated to an increased recurrence rate. Recurrence is attributed to various factors -4, for iTreatment modalities are varied, among the conservative options, surgical decompression is a popular technique, which creates a communication between the cystic cavity and the oral environment through a device in order to relieve the internal pressure within the cyst, and therefore, the size and volume of the lesion, allowing the preservation of adjacent vital structures -4. Additet al. .-Histopathology and immunohistochemistry Morphological changes of OKC were evaluated by H&E staining. Sections of 4µm were cut for indirect biotin-streptavidin-peroxidase staining. For antigen retrieval, Bulleye Decloaker  in a pressure cooker in microwave at a maximum pressure (750W) for 5 minutes was used. Tissue sections were incubated with Ki-67 , mini-chromosome maintenance protein (MCM) MCM4 , MCM7 , Bcl2  and polyclonal Bax  antibodies. Detection and amplification system Envision (DAKO®) was applied for 30 minutes. Slices were exposed to 3’-diaminobenzidine-H2O2 (DAKO®) and finally stained with Mayer’s hematoxylin.-Ki-67 and MCM4/7 cell quantificationNuclear immunoreactivities were assessed as described previously . A 40x m-Bax and Bcl-2 cell quantificationBax and Bcl-2 positivity was evaluated semi-quantitatively by cytoplasmic cell expression in the epithelial cyst. When percentage of immunopositive cells were equal to zero, it was cataloged as negative, less than 25% was categorized with one + (mild), between 25 and 50% with ++ (moderate) and, finally equal or greater than 50% with +++ (intense).-Statistical analysisp value was less than 0,05.Data obtained were analyzed using mean ± standard deviations (SD) in addition to frequencies and percentages. Quantitative variables were compared using Student t-test for related samples, assuming a normal distribution. Qualitative variables were compared using Wilcoxon test for paired samples. Comparisons were considered statistically significant if A total of 21 patients were included in this study, distributed in 13 women  and 8 men . The mean age was 27, 33±14, 9 years, with a minimum age of 9 years and a maximum age of 58 years.Of the 21 cases evaluated, most were found in the mandible, all 13 cases located in the posterior area , the rest were situated in the maxilla, 6 cases in the anterior area  and 2 in the posterior area . No cases were found in the anterior mandibular area.Between the samples taken, mean time of decompression was 13,14±16,68 months. The shortest decompression time applied was 1 month and the longest 60 months .-Histopathological changesp=0,007) (Loss of parakeratinization in some area of the cystic epithelium was observed before decompression (4 cases). However, after surgical decompression, only 8 cases  preserved a parakeratinized epithelium, while in 13 cases  there was loss of parakeratinization in various areas or in the entire cystic epithelium. Loss of superficial parakeratinization was statistically significant after surgical decompression  .Presence of atypical mitosis was only observed in one pre-decompression case. Epithelial mitosis was not observed in post-decompression cases.p=0,467)  and augmented in 9  cases. On the contrary, in the post-decompression samples we observed uniform thickness only in 5 samples , and epithelium hyperplasia in 11 . Absence of epithelium was observed in 5 cases . When results were compared, differences were not statistically significant  .p=0,002)  .p=0,029) . In contrast, all post-decompression cases showed inflammation   .-Cell proliferation p=0,323),  However, this reduction was not statistically significant , , . Again, decreased expression of MCM7 was not statistically significant , . 5 cases were negative. Similarly, after decompression treatment, 4 samples did not show any Bax staining and 17 cases were positive  . Bax resThe expression of Bcl-2 in the cystic epithelium was observed in 14 pre-decompression and 14 post-decompression samples. No changes were observed in Bcl-2 expression before and after decompression .OKC responds to conservative treatments such as surgical decompression, inducing modification of lining cystic wall until resemble normal oral mucosa ,9. The rDecompression time was arbitrarily determined by each clinician and varied widely in relation to the anatomical location of the cases. Interestingly it was observed that the longest time ranges were the cases located in the posterior mandibular area, this may be due to the fact that in this space, OKCs tend to reach larger dimensions without being noticeable or because the mandibular bone is denser, taking longer to regenerate, unlike the maxilla.OKC presents a morphology that confers histopathological characteristics that differentiate it from other neoplasms or developmental cysts, features associated with its recurrence and aggressiveness potential. Epithelial features of the OKCs can be altered by decompression due to intense inflammation, to the point that the histopathological identification of the lesion may become more complex or even impossible .et al. (et al. (Marker et al.  describe (et al.  establisPartial or total loss of parakeratinization, decrease in the basal distribution of palisade cells, epithelial hyperplasia and an increase in capsular fibrosis induced by inflammatory changes are histopathological changes that allow, in decompressed OKC, an easier separation of the cystic wall from the bone tissue in a second phase, reducing the possibility of recurrence associated with the permanence of the remaining cystic tissue within the surgical area and facilitating the final treatment -12.et al. (Cottom et al.  describeRegarding cell proliferation, a discrete decrease in the expression of Ki-67 and MCM4/7 was observed in OKC after surgical decompression in our study, suggesting a reduction of proliferative activity and therefore an apparently less aggressive behavior, probably by detection of aberrant cell growth and/or multiplication.et al. (et al. (et al. (Nakamura et al.  coincide (et al.  and Clar (et al.  found noet al. (et al. (To our knowledge there are no publications to date using of MCM4 or MCM7 as a cell proliferation marker in OCK before and after surgical decompression. Acharya et al.  and Jaaf (et al.  both rep (et al. .On the other hand, cell death can be assessed through the expression of the Bcl-2 family proteins, they have the ability to regulate the apoptosis process through pro and anti-apoptotic proteins such as Bax and Bcl-2 respectively. An increase in the detection of Bax favors cell death, while an excess in the expression of Bcl-2 prolongs cell survival by inhibiting apoptosis, promoting the development of a pathological lesion. The positivity of both markers in OKC has been previously reported independently, coinciding with a high expression of Bcl-2 that the authors correlate with the aggressive clinical behavior of the cyst -26.et al. (et al. (Soluk Tekkeşın et al.  evaluate (et al.  who alsoet al. (et al. (In contrast, Tenorio et al.  and Rang (et al.  reportedet al. (Study of Diniz et al.  is the oNo changes of Bax and Bcl-2 were observed in our study. The recovery or maintenance of the balance between pro and anti-apoptotic cells during or after surgical decompression, may suggest slowing of cyst growth, which with other factors contributes to its involution and therefore decompression success. To our knowledge, there are not previous studies that compare the expression of Bax in OKC before and after surgical decompression.OKC treatment should be aimed to those modalities that eradicate the lesion with a low risk of recurrence at a low morbidity rate -4, evaluSurgical decompression generates histological structural changes in OKC both in the epithelium and in the conjunctive wall generated by inflammation; however, these findings do not seem to alter the induction of the cell cycle or the rate of epithelial apoptosis.


Background: Multisite pain is commonly chronic and often lacks its initial role as a potential tissue damage signal. Chronic pain among working-age individuals is a risk for disability and imposes a major burden on health care systems and society. As effective treatments for chronic pain are largely lacking, better identification of the factors associated with pain over working years is needed.Methods: Members of the Northern Finland Birth Cohort 1966 participated in data collection at the ages of 31  and 46 . Using these two time points, we performed a multivariable analysis of the association of socioeconomic, occupational, psychological and lifestyle factors  with the number of musculoskeletal pain sites . The data were analyzed using generalized estimating equations.Results: At the age of 31, multisite pain was reported by 72.5% of men and 78.6% of women. At the age of 46, the prevalence of multisite pain was 75.7% among men and 82.7% among women. Among men, the number of pain sites was positively associated with age , low household income , unemployment , any occupational exposure , regular smoking , and psychiatric symptoms . Among women, the number of pain sites was positively associated with age , unemployment , any occupational exposure , regular smoking , overweight , and psychiatric symptoms ; living alone was negatively associated with the number of pain sites .Conclusion: Of the studied predictors, psychiatric symptoms, occupational physical exposures and unemployment were most strongly associated with multisite pain among both sexes. The results of this study deepen the understanding of the underlying factors of and comorbidities behind multisite pain, and help develop pain relief and rehabilitation strategies for working-age individuals with multisite pain. Pain is an unpleasant experience, traditionally perceived as a signal of actual or potential tissue damage . It is aThe study of multisite pain and its underlying factors has gained scientific interest internationally. Several studies suggest that multisite pain is more common among females than males –7 and amMost previous evidence is limited by its cross-sectional setting, although pain status and its determinants may fluctuate across adulthood. In addition, only a few studies have been able to simultaneously model a wide range of underlying factors including socioeconomic, occupational, psychological and lifestyle dimensions. It is important to account for all these dimensions together in a comprehensive dataset in order to evaluate their interrelations and reveal independent effects on an individual's pain status across adulthood. Finally, prospective studies with large population-based samples are essential for reaching high general representativeness. Studies focusing on working-age individuals should be of particular importance from the perspective of economic impact and timely rehabilitation.In this study, we aimed to evaluate the association between multidimensional risk factors and the number of musculoskeletal pain sites at two timepoints in adulthood. As previous studies have revealed several underlying factors of multisite pain, we aimed to perform a comprehensive multivariable analysis, in an attempt to identify individual predictors of pain status from the pool of socioeconomic, occupational, psychological and lifestyle dimensions. With our data, we were also able to compare the identified predictors in terms of effect size. In accordance with previous studies, we hypothesized that the number of pain sites would be associated with older age, indicators of lower socioeconomic status, the presence of occupational exposures, unhealthier lifestyle, obesity, and psychiatric symptoms.n = 4,028 respondents). In 2012–2014 when the cohort members were 46 years old, they received a new questionnaire enquiring about the same variables as before . At both time points, the questionnaires included the same questions on education, marital status, household income, employment, occupational physical exposures, physical activity, smoking status, alcohol use, psychiatric symptoms, and musculoskeletal pains. In addition, the cohort members' height and weight were measured at both times, as part of clinical examinations. All observations were included, i.e., individuals were not excluded if they had data from only 31 or 46 years. The outline of the study is illustrated in This study was based on the Northern Finland Birth Cohort 1966 (NFBC1966) population , which iAge was calculated on the basis of birth dates. However, as the sample was a 1966 birth cohort and thus coeval, the age variable was dichotomous .Basic education was reported on the basis of the following alternatives: (1) Uncompleted compulsory school, (2) Compulsory school, (3) Completion of matriculation examination. Additional education was reported on the basis of the following alternatives: (1) None, (2) Occupational course, (3) Vocational school, (4) Other lower-level institute/academy/college, (5) Polytechnic, (6) University, (7) Other, (8) Not yet completed. Low education was defined as having only compulsory school .Marital status was selected from the following alternatives: (1) Married, (2) Cohabiting, (3) In a registered partnership, (4) Unmarried, (5) Divorced, (6) Divorced from a registered partnership, (7) Widow, (8) Widow from a registered partnership. Individuals who were not married, in a registered partnership or cohabiting, were considered to live alone.Household income was elicited by asking: “Please report the total income of your household last year (before taxes).” The respondents were also asked to report the number of family members in their household. Household income (per household member) less than the participants' median value at each time point was defined as low household income .Employment status was selected from the following alternatives: (1) Permanent full-time employee, (2) Permanent part-time employee, (3) Temporary full-time employee, (4) Temporary part-time employee, (5) Full-time self-employed or entrepreneur, (6) Part-time self-employed or entrepreneur, (7) Full-time student, (8) Part-time student, (9) Unemployed for <6 months, (10) Unemployed for 6–12 months, (11) Unemployed for > 2 months, (12) Employed/educated through labor market support, (13) Laid off or reduced working hours, (14) Maternity/paternity leave or parental leave, (15) Retired, (16) Caring for my own household, (17) Other. Individuals who were not working or studying full-time or part-time, self-employed or entrepreneurs, employed/educated by labor market support, or on parental leave, were considered unemployed .The presence of several occupational physical exposures  were each elicited separately with the following response alternatives: (1) Never or very rarely, (2) Rarely, (3) Occasionally, (4) Often, (5) Very often. Individuals who reported encountering any of the enquired exposures at least often were considered to have an occupational exposure . We alsoLeisure-time physical activity was elicited by the following question: “How often do you engage in brisk exercise (getting out of breath and sweating)? (1) Once a month or less, (2) 2–3 times a month, (3) Once a week, (4) 2–3 times a week, (5) 4–6 times a week, (6) Daily.” Individuals who reported exercising less than once a week were defined as inactive .Smoking habits were elicited by the following question: “Do you currently smoke? (1) 7 days a week, (2) 5–6 days a week, (3) 2–4 days a week, (4) Once a week, (5) Occasionally, (6) Not at all.” Alcohol consumption was reported by answering the following questions: “Do you consume alcohol, even occasionally (yes/no)?”; “How often do you consume alcohol ? (1) Never, (2) Once a year or less, (3) 2 times a year, (4) 3–4 times a year, (5) Once in 2 months, (6) Once a month, (7) 2–3 times a month, (8) Once a week, (9) Several times a week, (10) Daily.” Individuals who reported smoking or drinking more often than once a week were considered regular smokers and drinkers, respectively. While there are no universal definitions for regular smoking  or drink2 , with the participant barefoot and in their underwear. At the 46-year follow-up, height was measured twice, and the mean of two measurements was used as the final height. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared, and overweight was defined as BMI ≥ 25 kg/m2 .Symptoms of anxiety and depression were screened using the validated Hopkins Symptom Checklist-25 (HSCL-25)  total meThe presence of musculoskeletal pain during the previous year was elicited separately for four anatomical areas: the neck-shoulder region, lower back, upper extremity, and lower extremity  and the outcome , we used a generalized estimating equations (GEE) approach. GEE is a regression-based method which is particularly suitable for the analysis of longitudinal or otherwise correlated data, as it accounts for all time points and is able to correct for correlations between variables by introducing a working correlation matrix . The threshold for statistical significance was set at n matrix , 31. We P-values were recorded from the GEE output. RRs represent the relationship between a predictor variable and the outcome over the follow-up.First, we evaluated the univariate associations between each predictor and the outcome. Then, as our main analysis, we constructed full multivariable models which included all the predictors. Finally, we ran a sub-analysis, which specified the independent roles of each occupational exposure relative to the outcome. All variables except for sex were treated as time varying. As we expected to encounter clear sex differences in the predictors of pain status, we stratified all models by sex. All observations were included, i.e., individuals were not excluded if they had only responded at 31 or 46 years, under the assumption of data missing at random. Exponentiated regression coefficients , 95% Wald confidence intervals (CIs), and The study followed the Declaration of Helsinki principles and was approved by the Ethical Committee of the Northern Ostrobothnia Hospital District together with the Scientific Committee of the Northern Finland Birth Cohorts. The study was based on voluntary participation and the cohort members gave their written informed consent to use their data in scientific studies at both time points. The data were processed anonymously.n = 2,026 and n = 1,589 at the ages of 31 and 46, respectively) and 3,842 observations of women . Our analysis was based on a total of 3,615 observations of men  was the strongest predictor of multisite pain among both sexes (RR 1.21 among men and 1.19 among women). It is evident that psychological and psychiatric factors such as depression, anxiety, catastrophic thinking, and fear of movement play an important role in the transition to chronic pain and disability . The preOur data underline the important role of socioeconomic and occupational determinants in multisite pain. Among both men and women, unemployment was independently associated with the number of pain sites (adjusted RR 1.13 among men and 1.10 among women), while household income had an independent association with the number of pain sites only among men. Our multivariable analysis thus provides further evidence of the independent negative role of unemployment in multisite pain . EngagemPrevious studies have associated multisite pain with smoking, physical inactivity and obesity , 11, 16.The biopsychosocial perspective to medicine, as expedited by Engel , has beeThe main strength of this study was that it was based on a large general population birth cohort covering all sectors of the economy and occupations. Since all the participants were of the same age, any bias originating from macroeconomic fluctuations were avoided. The two-timepoint setting with data from the ages of 31 and 46 covers the phase of the life course during which one is expected to be fit for gainful work; from this perspective, using our results to further develop care and rehabilitation among individuals with multisite pain has notable economic potential. The study population is well-known, and we had access to a wide range of variables across their adulthood. As such, we were able to aggregate a wide variety of biopsychosocial variables accompanied by detailed variables of economic situation and various occupational physical loads in a set of models, in which the effects of their mutual correlations on the results could be accounted for and the independent effects of the variables could be identified. We ran separate models for men and women in order to study the predictors of multisite pain among each sex separately.This study also had limitations. The questions regarding pain were in the dichotomous format of yes/no. Unfortunately, we did not have data on the intensity, frequency, bothersomeness, disability, or other dimensions of pain from the full course of the follow-up, and we were unable to supplement our dataset with these missing details in a retrospective manner. We also modeled the explanatory factors using dichotomous classifications in order to allow direct comparisons of effect size in terms of RRs. Finally, we had no data on pain or explanatory factors from the interval between the ages of 31 and 46. Except for BMI, all variables were self-reported, which may increase the risk of response bias in our data.In conclusion, we identified several independent predictors of multisite pain among working-age Finns of Northern origin. Among men, these were age, low household income, unemployment, occupational exposures, regular smoking, and psychiatric symptoms. Among women, they were age, living alone, unemployment, occupational exposures, regular smoking, overweight and psychiatric symptoms. It remains to be seen whether the same set of predictors can be identified in younger or older populations and whether the predictors are age dependent. Experimental studies are needed to improve and test interventions targeted at the main predictors identified by the present study to decrease the prevalence of potentially disabling multisite pain among working-age individuals.via electronic material request portal . In the use of data, we follow the EU general data protection regulation (679/2016) and Finnish Data Protection Act. The use of personal data is based on cohort participant's written informed consent at his/her latest follow-up study, which may cause limitations to its use. Please, contact NFBC project center (NFBCprojectcenter@oulu.fi) and visit the cohort website (https://www.oulu.fi/nfbc) for more information. Requests to access the datasets should be directed to University of Oulu, Infrastructure for Population Studies, NFBCprojectcenter@oulu.fi.The datasets presented in this article are not readily available because The Northern Finland Birth Cohort 1966 data is available from the University of Oulu, Infrastructure for Population Studies. Permission to use the data can be applied for research purposes The studies involving human participants were reviewed and approved by Ethical Committee of the Northern Ostrobothnia Hospital District. The patients/participants provided their written informed consent to participate in this study.V-HA, VR, LA-M, JK, and PO were involved in data collection. V-HA and PO analyzed the data. V-HA wrote the manuscript draft and prepared the figures. All authors read and approved the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


COL4A5 gene. We were only able to make the final diagnoses in each of the family members after genetic testing and reverse phenotyping. This case highlights the utility of genetic testing and reverse phenotyping in resolving clinical diagnosis in families with unusual constellations of different glomerulopathies. We propose that clustering of different glomerular disease phenotypes in a family should be an indication for genetic testing followed by reverse phenotyping.Glomerular diseases (GDs) are a major cause of chronic kidney disease in children. The conventional approach to diagnosis of GDs includes clinical evaluation and, in most cases, kidney biopsy to make a definitive diagnosis. However, in many cases, clinical presentations of different GDs can overlap, leading to uncertainty in diagnosis and management even after renal biopsy. In this report, we identify a family with clinical diagnoses of postinfectious glomerulonephritis and IgA nephropathy in a parent and two children. Renal biopsies were initially inconclusive; however, genetic testing showed that the two individuals diagnosed at different points with IgA nephropathy carried novel segregating pathogenic variants in  COL4A3, COL4A4, and COL4A5) (Glomerular diseases (GDs) account for 8–14% of all cases of chronic kidney disease (CKD) in children . The patCOL4A genes, and pathogenic variants in these same genes have also been reported in patients with presumed IgA nephropathy  are due to pathogenic variants in hropathy –10. ThisYB is a 7-year-old male with no significant medical history who presented to the emergency department with hematuria in the context of 3-day history of fever, cough, and sore throat. He denied dysuria, epistaxis, hematochezia, or mucosal bleeding. Physical examination was notable for normal blood pressure, normal oropharyngeal appearance, and absence of peripheral edema. Urinalysis revealed 1+ protein, 3+ blood, and 1+ leukocytes. Urine protein/creatinine ratio was 1,167 mg/g. Serum creatinine was 0.5 mg/dl. Additional serum chemistries, complete blood count, C3/C4, anti-streptolysin O, and anti-DNase B were all within normal limits. Serologic testing was negative for anti-dsDNA, ANCA, and anti-GBM. Renal ultrasound revealed symmetrically large kidneys bilaterally without hydronephrosis, nephrolithiasis, or masses. He was treated for presumed UTI and referred to a pediatric nephrology clinic for follow up.Approximately 2 months following his initial presentation, YB had recurrence of gross hematuria concomitantly following a viral syndrome. Rapid streptococcal testing was negative. Urinalysis revealed 3+ blood and persistent subnephrotic-range proteinuria. His symptoms resolved within a week. Given the concern for postinfectious glomerulonephritis vs. IgA nephropathy, close follow-up was arranged. Over the subsequent 9 months, he continued with microscopic hematuria and low-grade proteinuria. Upon further review of family history, it was discovered that his mother had microscopic hematuria and hypertension at 9 years of age, which was thought to be due to IgA nephropathy; she was managed conservatively, and his maternal great grandfather had a history of end-stage kidney disease (ESKD) of unclear etiology . Given pCOL4A and FSGS genes. Results revealed two variants of uncertain significance (VUS) in CD2AP  and COL4A5  database. The variant was predicted to be damaging by three in silico tools. The CD2AP variant was found in 14 individuals in the gnomAD database  and was also predicted to be benign by SIFT in silico tools. Based on these findings, immunofluorescent staining for COL4A was performed on the previously obtained kidney biopsy and demonstrated negative staining for collagen type IV alpha 3 and 5 in the glomerular basement membranes, negative staining for alpha 5 in Bowman's capsule, and positive staining for alpha 2 in the glomerular basement membranes and mesangium, all findings consistent with COL4A disease  presented to the emergency department (ED) with a 2-day history of fever, poor oral intake, emesis, abdominal pain, and oliguria. History was significant for Group A streptococcal pharyngitis diagnosed 1 month prior. Physical examination in the ED was notable for hypertension, petechial rash on the face and chest, and bilateral pitting pedal edema. Renal function panel revealed hyperkalemia (7.1 mmol/l) and stage three acute kidney injury . Urinalysis revealed 3+ blood, 2+ protein, urine protein/creatinine ratio of 388 mg/g, low C3 complement, and normal C4 complement. Serology and infectious disease work up were all negative. A presumptive diagnosis of postinfectious glomerulonephritis was made, but a renal biopsy was performed to rule out Alport syndrome in view of the family history. Histology of the kidney biopsy done on OB showed diffuse proliferative and exudative glomerulonephritis with global finely granular and focal segmental globular IgG and C3 deposits on immunofluorescence. Electron microscopy showed extensive subendothelial immune complex deposits as well as occasional subepithelial “hump-like” deposits, consistent with postinfectious glomerulonephritis . Alport  brother .This case highlights the utility of genetic testing and reverse phenotyping in resolving clinical diagnosis . While pCOL4A3, COL4A4, and COL4A5) (COL4A5), and the remaining 15% of mutations are inherited in an autosomal recessive or autosomal dominant manner (COL4A3 or COL4A4 mutations) (Alport syndrome (AS) is an inherited glomerulopathy characterized by hematuria, proteinuria, progressive renal failure, hearing loss, and ocular abnormalities . Alport  COL4A5) . 85% of tations) , althougA 2018 and, more recently, a 2021 consensus report suggests new classification for AS because of wide variability in its clinical phenotype and estimated that the lifetime risk for chronic kidney disease (CKD) is 100, 25, 100, and 20% for X-linked recessive, X-linked heterozygous, autosomal recessive, and autosomal dominant AS respectively .COL4A-associated nephropathy and significant overlap of this condition with other distinct glomerular pathologies. We and others reported that up to 10% of patients with diagnosis of familial FSGS have pathogenic variants in one of the COL4A genes; subsequent large studies showed that COL4A variants are the most common cause of CKD of unknown etiology in adults . However, further clinical evaluation and additional ancillary work-up clearly establish the pathogenicity of this variant. In addition, it also allows for accurate diagnosis and management of a sibling who presented with a completely different non-monogenic glomerular disease shortly after establishing diagnosis of AS in the proband. Our findings highlight the need for the future nephrologists to be trained in the use and interpretation of genetic testing to make accurate diagnosis of different kidney diseases. They also emphasize the need for integration of clinical, pathologic, and genomic data in the diagnosis of glomerular and other kidney diseases.The In conclusion, we propose that unusual aggregation of different glomerular diseases in a family is an indication for genetic testing followed by reverse phenotyping.RG design the studies, participate in the recruitment of participants in the study, genetic studies, and deep phenotyping, and writing of the manuscript. RK participate in the recruitment of participants in the study, genetic studies, and deep phenotyping and writing of the manuscript. VK participate in the recruitment of participants in the study, deep phenotyping and editing of the manuscript. MS performed the genetic studies and was involved in writing of the manuscript. BL performed the genetic studies and was involved in writing of the manuscript. AB, LB, and DH generated and interpreted the renal biopsy. All three were involved in writing and editing of the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.