Background and Objectives: Autoimmune bullous diseases (AIBDs) may be treated with intravenous immunoglobulin (IVIG) infusions. This study aimed to evaluate the benefits and safety profiles of high-dose IVIG therapy in AIBD patients, as determined by clinical remission, the glucocorticosteroid-sparing effect, and adverse events at 12 months follow-up in a Central European university dermatology department setting. Materials and Methods: Our case series included 10 patients: five patients with pemphigus vulgaris, one with pemphigus herpetiformis, one with pemphigus foliaceus, one with bullous pemphigoid, two with epidermolysis bullosa acquisita. They underwent 4–12 monthly cycles of IVIG therapy at a dose of 2 g/kg per cycle. Results: The prednisone dosage reduction after 2, 6, and 12 months following the final IVIG course was 65.45%, 70.91%, and 76.37%, respectively. During the 12-month observation period, disease relapse was observed in 20% of patients, while others achieved complete or partial remission without or with minimal therapy. Side effects were seen in 80% of patients; they were transient and did not necessitate discontinuation of IVIG. Conclusions: IVIG demonstrates effectiveness as a treatment with a favorable safety profile. Nevertheless, its high cost remains a significant drawback, particularly in low-income countries. IVIG should be considered, especially in patients opposed to standard therapies or with contraindications to their use. Autoimmune bullous diseases (AIBDs), of which bullous pemphigoid (BP) is the most common in Europe, are a group of relatively rare, potentially life-threatening diseases caused by autoantibodies targeting adhesion molecules in the skin and/or mucous membranes, resulting in varying inflammatory responses . AIBDs, Systemic glucocorticosteroids (GCS) remain the first-line, live-saving drugs in pemphigus; however, the high doses and prolonged courses required for disease control can lead to many dangerous side effects, including death . AchieviHuman immunoglobulin has been used to treat people since 1952 when Bruton treated a child with recurrent pneumococcal infections, followed by Imbach et al. who used IVIG to treat a child with immune thrombocytopenia ,8. Due tAdding IVIG to the standard therapy of PV  seems to lead to long-term remission without drugs, achieved through its ability to eliminate selected disease-specific IgG antibodies ,13. MoreThis study aimed to evaluate the effectiveness of the treatment of autoimmune bullous diseases with intravenous immunoglobulins during a 12-month observation period.-Patients who were receiving a prednisolone equivalent dosage of less than 10 mg/day prior to the first IVIG cycle, except for those with absolute contraindications to GCS,-Patients who had fewer than 10 active lesions  before the initial IVIG infusion,-Patients for whom follow-up data was unavailable for at least one year after the completion of the last IVIG cycle,-Patients with a history of hypersensitivity reactions to the tested drug,-Patients diagnosed with IgA deficiency.The medical records of patients diagnosed with AIBDs who underwent IVIG treatment at the dermatology department in Poznan between 2014 and 2022 were retrospectively analyzed. The study employed the following exclusion criteria:Following the application of these exclusion criteria, a total of 10 patients met the inclusion criteria and were included in the study.The diagnoses of PV, PF, BP, EBA, and pemphigus herpetiformis (PH) were based on clinical signs, histological findings, and immunological studies: direct immunofluorescence (DIF), indirect immunofluorescence, and multiplex ELISA for IgG antibodies to desmoglein 1, desmoglein 3, BP 180, BP 230, envoplakin, and type VII collagen .Patients included in the study were treated with IVIG infusions intravenously for at least four consecutive courses, with a maximum of twelve courses. The total dose of 2 g/kg per cycle was divided into five equal doses (400 mg/kg) and administered on five consecutive days in a hospital setting. Each cycle was repeated every 4 weeks. Blood pressure and urine production were monitored during each infusion. Before each cycle, a complete blood count, renal and liver function tests, and urine analysis were performed. Before the first cycle, serum immunoglobulin levels were also measured. The patients were followed up 2, 6, and 12 months after their last IVIG course.We used the consensus described by Murrell DF et al. to define the control of disease activity . A complIn total, 10 patients  were included in our case series study. There were five females and five males aged 58.5 ± 14.7 (mean ± SD) years, with an age range of 30 to 78 years. The time from disease diagnosis to the start of IVIG therapy was a minimum of 0.3 years and a maximum of 6.5 years, with a mean of 1.76 years ± 1.85.General patient characteristics are shown in The mean initial prednisone equivalent dosage was 30.56 mg ± 21.14, and it was gradually tapered in patients with controlled disease. Following the first two IVIG cycles, the prednisone equivalent dosage was reduced by approximately half, and subsequent tapering continued until the cessation of IVIG treatment. However, some patients required an increase in dosage to effectively manage the disease. Two months after completing all IVIG cycles, the mean prednisone equivalent dosage was 10.56 mg ± 4.9. At 6 months, it further decreased to 8.89 mg ± 6.5, and at 12 months, it reached 7.22 mg ± 7.49. Therefore, the reductions in dosage were 65.45%, 70.91%, and 76.37% at 2, 6, and 12 months, respectively.Prior to the administration of IVIG therapy, patients were treated with oral GCS (OGCS), various immunosuppressants, RTX, and DP. Some patients required additional therapy after IVIG infusions. Patient #4, a 38-year-old with PV, received DP 50 mg/day, while patient #7, a 56-year-old female with PH, was taking AZA 50 mg/day. Patient #9, a 60-year-old female with EBA, had two infusions of RTX 1 g each and received methylprednisolone pulse intravenously (1 g daily over 3 days).In our calculations of prednisone equivalent dosage, we excluded one patient with PV (#1)  who, dueIn the case of patient #7 with PH, IVIG proved to be a life-saving procedure. The patient experienced exacerbation of her disease, triggered by an incarcerated Meckel’s diverticulum, which led to bowel obstruction and feculent vomiting. Patient #9 with EBA experienced a direct exacerbation of the disease involving mucosal tissues immediately after IVIG therapy, resulting in the need for a tracheostomy. Despite receiving RTX infusion, there was no improvement observed at the 3-month follow-up. As a result, the patient received methylprednisolone pulse (1 g daily over 3 days), which eventually brought the disease under control.We also encountered a patient with severe PV specifically involving the conchae of the auricles. This patient underwent five IVIG cycles, which resulted in complete remission of PV symptoms. However, due to the unavailability of a comprehensive 1-year follow-up period, it was not feasible to analyze further this patient according to our study design.The treatment effects of the aforementioned patient and patient #4 are depicted in Initially, following the first treatment cycle, patients exhibited notable clinical improvement. However, over the subsequent 2 months of treatment, a decline in treatment response became evident. Specifically, relapse of the disease was observed in 50% of patients at the 2-month mark, which subsequently decreased to 20% after a 12-month period. Additionally, at the 2-month evaluation, 10% of patients achieved complete remission with no treatment (CRNT), while the number increased to 20% during the 12-month follow-up period. Partial remission with no treatment (PRNT) and complete remission with minimal treatment (CRMT) were observed in 40% and none of the patients, respectively, at the 2-month observation, but these percentages changed to 50% and 10%, respectively over time. Data about patients’ response to IVIG therapy (first five cycles and 1-year follow-up) is shown in Side effects were observed in 80% of the patients, demonstrating a transient nature that did not warrant the discontinuation of IVIG therapy. Among these, the most prevalent side effect was leukopenia, which manifested after the first infusion in patients #1 and #5, after the third cycle in patient #3, and following the seventh infusion in patient #10. During the initial IVIG infusion, three patients  experienced hypotension, prompting a reduction in the infusion rate. Similarly, in the case of two patients (#2 and #8) who experienced headaches after their first IVIG cycle, the infusion rate was accordingly decreased. Over the 1-year follow-up period, two patients (#3 and #8) developed fungal infections in the oral cavity, which were effectively managed using systemic antimycotics. Furthermore, patient #10 reported experiencing nausea shortly after the second infusion, which spontaneously resolved within a few hours. Patients’ treatment characteristics are shown in This study was subject to certain limitations, including a small sample size. The rarity of AIBDs and the high cost associated with IVIG therapy contribute to the limited availability of participants. Additionally, the heterogeneity of our patient group, consisting of individuals diagnosed with various AIBDs, poses a challenge. However, it should be noted that IVIG treatment promotes the accelerated degradation of pathological autoantibodies produced during the course of various AIBDs, thereby exhibiting a similar mechanism of action regardless of the specific diagnosis . FurtherIn 2009, Amagai et al. conducted the first randomized double-blind trial, demonstrating the efficacy of high-dose IVIG in the therapy of pemphigus. In 2017, the same authors designed a similar study in patients with BP, reaching the same conclusion . In our Of our participants, 80% were able to maintain partial and complete remission at 12 months, of whom 60% required minimal therapy. This long-term remission is likely associated with IVIG’s ability to modulate the function of Fc receptors and affect the production of pathological autoantibodies ,19. MoreFurthermore, in 2016 Enk et al. promoted IVIG as second-line therapy in AIBDs refractory to standard therapy. They also recommended considering IVIG in patients previously treated with RTX in whom disease control was not attained . One patDue to the rarity of EBA, there is no specific treatment dedicated to this disease. IVIG was proposed by Miyamoto et al. as part of its therapy . SesarmaAdverse reactions accompanying IVIG infusions occur, on average, in 20% of patients, and the majority of them are mild . Flu-likLeukopenia emerged as the most frequently observed side effect in our patient cohort. According to Cicha et al., out of 35 patients who received either low or high doses of IVIG, a decrease of ≥10% in absolute leukocyte counts was observed in 30 individuals. Among the factors implicated in the occurrence of leukopenia following IVIG administration, two mechanisms can be discerned: the production of antibodies targeting neutrophils and the formation of immunoglobulin aggregates, which modulate the function of surface receptors on neutrophils, triggering oxidative burst and degranulation, resulting in a decrease in the number of circulating cells . In our In conclusion, IVIG therapy was able to maintain a 1-year remission in patients with various AIBDs refractory to first-line treatment, however, it was not efficient in all cases. The safety profile of IVIG is good, as the therapy induced only transient adverse effects which were not the reason for the cessation of therapy. It seems to be beneficial to administer more IVIG courses to older patients, as their response to therapy may be weaker. High cost and limited availability, particularly in low-income countries, are the main disadvantages of IVIG therapy. Nevertheless, our case series study suggests that IVIG can be taken into consideration for the treatment of AIBDs, especially considering its favorable safety profile, even in low-income countries.


Case studies of rare oncologic metastases are an important source of clinical data for health care professionals and researchers. While infrequent, the knowledge base and clinical recommendations derived from such cases aid in advancements in the field. As such, we aim to add five cases to the growing body of literature. The first two male patients, aged 69 and 73, were diagnosed with colon adenocarcinoma, suspected to be a second primary prostate carcinoma, following positron emission tomography-computer tomography (PET-CT). This suspicion was ruled out by prostatectomy and histopathological investigations, which instead found adenocarcinoma of colonic origin. The next two male patients, ages 63 and 68, were diagnosed, respectively, with metastatic pancreatic adenocarcinoma with cardiac metastases and metastatic melanoma with distant metastases to the pancreas. The final patient was a 73-year-old male diagnosed with metastatic breast cancer after a radiological investigation of suspected renal cell carcinoma. Intriguingly, this risk surpasses that of developing a second primary colorectal cancer. According to some research findings, this phenomenon is particularly evident among male patients below the age of 65 [Cancers affecting the colon, rectum, and anus are commonly grouped under the term “colorectal cancer” in medical literature to ensure consistency. From a global perspective, the prevalence of colorectal cancer ranks third, after lung and breast cancers, in terms of incidence. With respect to fatalities; however, colorectal cancer takes a more significant toll, second only to lung cancer as the leading contributor to global cancer-related deaths . Men diage of 65 ,3. Men wge of 65 . Approxige of 65 . Collectge of 65 . The livge of 65 . Presentge of 65 ,8. Neverge of 65 ,11,12,13p = 0.0445) [Pancreatic cancer, while less frequent than colorectal cancer, is considerably more pernicious and lethal on a case-by-case basis. In fact, according to recent trends, the incidence and mortality rates of pancreatic cancer are predicted to increase in the following decades. Specifically, pancreatic cancer deaths surpassed those of breast cancer in 2016 and are expected to become the second-highest cause of cancer death by the year 2030 ,15. Aden 0.0445) .The amorphous nature of pancreatic tumors, growing around major vessels, generally makes resection impossible without prior radiation or chemotherapy . Only 10Metastatic melanoma has shown responsiveness to immunotherapy, as increased immune surveillance allows for a more robust inflammatory potential in the tumor microenvironment. This phenomenon is explained by inhibition of the innate immune-suppressive qualities of malignant cells, as compared to the limited reactivity of pancreatic adenocarcinoma, a so-called “cold” tumor . The immBreast cancer is extremely uncommon in male patients, accounting for around 1% of all cancers in men and less than 1% of all breast cancer cases . Owing tAn analysis of existing literature revealed a mere five documented cases involving patients with colon cancer that had progressed to prostatic metastasis. Notably, the earliest and most recent instances of such occurrences were diagnosed and reported in 1993 and 2013, respectively ,11,12,132, leucovorin 400 mg/m2, and 5-FU 400 mg/m2 on day 1, followed by a continuous IV infusion of 5-FU 2400 mg/m2 over 46 h every two weeks as adjuvant therapy for six cycles. One month after completing this treatment, the patient underwent exploratory laparotomy, in which no malignancies were found.The patient is a 69-year-old male with a 35 pack years (PY) smoking history and hyperlipidemia who presented with abdominal pain and melena. He has no family history of cancer. In October 2015, he was diagnosed with stage 4 colon adenocarcinoma (T3 N2 M1) with peritoneal metastasis. In November 2015, the patient underwent a left hemicolectomy and further histological investigation of a subdiaphragmatic nodule. He began chemotherapy under the FOLFOX protocol with the inclusion of bevacizumab (Avastin). Specifically, the intravenous (IV) regimen incorporated bevacizumab 5 mg/kg, oxaliplatin 85 mg/mThe patient was examined two years later (April 2017). PET-CT showed areas of hypermetabolic uptake in the liver (1.7 cm in diameter), right colon, left iliac lymph-nodes, and prostate .A second primary prostate cancer was suspected upon further investigation. As a result, prostate-specific antigen (PSA) was measured and found to be elevated at 6.2 ng/mL . Magnetic resonance imaging (MRI) of the pelvis showed a mass over the seminal vesicle, and prostate cancer could therefore not be excluded .The patient underwent a prostatectomy with histopathological analysis showing adenocarcinoma of colonic origin. The patient is currently undergoing intermittent chemotherapeutic treatment (August 2023).The patient is a 73-year-old male with a 25 PY smoking history ischemic heart disease, diabetes mellitus, chronic kidney disease, hypertension, and hyperlipidemia. He denied any family history of cancer. The patient complained of constipation and abdominal pain, warranting an investigative analysis of the abdominal region. In September 2009, he was diagnosed with stage 3 adenocarcinoma of the sigmoid colon (T3 N1 M0).2, leucovorin 400 mg/m2 and 5-FU 400 mg/m2 on day 1, followed by continuous IV infusion of 5-FU 2400 mg/m2 adjuvant therapy over 46 h every two weeks) for six cycles. In December 2014, PET-CT showed hypermetabolic uptake in the area of the prostate .The patient is a 63-year-old male with a 35 PY smoking history, dyspnea, and dyslipidemia. He has no family history of cancer. As a result of shortness of breath, a CT angiography of the chest was performed, which showed pulmonary embolisms in both lungs. A CT of the chest and abdomen showed a mass in the head of the pancreas, secondary growth patterns in the liver, and pulmonary emboli in the segmental branches of the pulmonary arteries. A consolidation in the right lower lung appeared to be the result of an infarct.A liver biopsy performed under ultrasound guidance showed metastatic adenocarcinoma of unknown primary origin. However, consideration of the pancreato-biliary system was warranted. Laboratory results showed elevations in liver enzymes  and no increase in bilirubin. Cancer markers were also elevated, namely CA-125 (2072), CA-19-9 (386034), and CEA (471). Due to left chest pain, an echocardiogram was performed, which showed normal left ventricular systolic function, preserved right ventricular function, mild tricuspid regurgitation, and severe pulmonary hypertension.As part of further investigation into a likely malignancy, PET-CT showed a hypermetabolic region in the head of the pancreas indicative of a primary tumor. Additionally, there was a hypermetabolic mass between the pancreas and the third part of the duodenum. Finally, metastases were identified via increased hypermetabolic activity throughout the liver and portacaval lymph nodes. A distant bone metastasis was likewise discovered in the front part of the fourth rib with sclerotic changes. The apical myocardium was also found to have a possible metastasis from the same scan.Therefore, the patient underwent an echocardiograph, which revealed a mass in the right ventricle of the heart with a diameter of 4.3 cm × 3.5 cm . Based o2, leucovorin 400 mg/m2 and 5-FU 400 mg/m2 on day 1, followed by a continuous IV infusion of 5-FU 2400 mg/m2 over 46 h every two weeks) was ultimately attempted. After two cycles of FOLFIRINOX treatment, echocardiography of the heart was performed for follow-up, which showed a decrease in the diameter of the heart mass to 1.8 cm × 1.6 cm , hypertriglyceridemia, lumbar disc hernia, inguinal hernia, and depressive disorder. For these conditions, he was appropriately being treated with aspirin, omeprazole, atorvastatin, escitalopram, tamsulin hydrochloride, lorazepam and ramipril. The patient does not smoke or drink alcohol.The patient presented with a cutaneous mass in his groin, which was misdiagnosed as an epidermal cyst. He followed up with complaints of a growing mass in his groin, which was later found to be an enlarged lymph node. As a result, further evaluation was warranted. A CT of the abdomen showed enlarged lymph nodes and a possible tumor in the pancreas. Further CT showed nodules in the right lower lung (which were suspected to be a metastasis), a mass in the head of the pancreas (which were was suspected to be a primary tumor), and diffuse retroperitoneal, left groin and pelvic masses up to 2.5 cm in diameter. A biopsy under endoscopic ultrasound was performed on the pancreatic mass, which showed metastatic malignant melanoma .In addition, an excisional biopsy of the left groin lymph node showed metastatic melanoma. Therefore, the diagnosis of metastatic melanoma with malignant tumors in the lung, pancreas, and lymph nodes was appropriate. Treatment was performed according to ECOG guidelines. Specifically, he was given the first round of immunotherapy (ipilimumab 3 mg/kg plus nivolumab 1 mg/kg), which was well tolerated with minor complaints.This 73-year-old male patient has relevant disease history and risk factors. He was a cigarette smoker (20 PY) and had been treated appropriately for hypertension, hypothyroidism, ischemic heart disease and atrial fibrillation. He had a family history of cancer as well, as his mother was diagnosed with colon cancer at age 80, his sister was diagnosed with breast cancer at age 50, and two nephews were diagnosed with lymphoma at ages 8 and 15, respectively.The patient was also diagnosed with glomerulosclerosis in 2004. He therefore underwent a right kidney transplant in 2012, which failed, and a left kidney transplant in 2013, which succeeded.In November 2018, he presented with complaints of hematuria and subsequently underwent an abdominal CT, revealing a mass in the upper pole of the left kidney (4.7 cm × 5.4 cm). As part of further investigation, the patient underwent CT urography that confirmed the location of the mass in the upper pole of the left kidney, as well as blood clots in the renal pelvis and proximal urethra, bilateral pleural effusions, and traces of free fluid in the abdomen and pelvis . Renal cHistopathological analysis of the excised mass was highly suggestive of a breast tissue origin. Analysis of the hormone receptor status of the tumor resulted in a designation of estrogen and progesterone receptor as positive but human epidermal growth factor receptor 2 (HER-2) as negative. Ultrasound and mammography of the left breast showed prominent breast tissue throughout (BIRADS 2) , with siGiven the importance of statistics in medicine, case studies of rare metastatic cancer have value for scientists and medical professionals. We presented rare oncological metastases from five patients .The first two patients were diagnosed with colonic adenocarcinoma, with follow-up PET-CT demonstrating areas of hypermetabolic activity in the prostate. These prostate tumors were presumed to be secondary cancers. However, histopathologic analysis found them to be metastatic tumors of colonic origin. One of the patients initially refused prostatectomy and instead requested treatment according to the prostate cancer protocol. However, after consideration, he agreed to surgical intervention. Of note were the inconsistent PSA findings between these patients. Namely, neither had continuing metastatic pancreatic disease, although one patient (patient 1) had an elevated PSA of more than twice the upper limit of normal, while the other patient (patient 2) had levels within the normal range. One possible explanation is that PSA levels tend to rise with factors such as age, infection, trauma, and inflammation. Additionally, benign prostatic hyperplasia (BPH) can lead to increased serum PSA levels, which can reduce the reliability of this biomarker for predicting prostate cancer. Studies have indicated that as many as 86% of individuals with BPH may exhibit elevated serum PSA levels. Interestingly, research has revealed that PSA is expressed not only in prostatic tissues but also in various non-prostatic tissues. As a result, there is growing interest in exploring its potential as a biomarker for cancers beyond those of the prostate. Notably, elevated PSA levels have been associated with colorectal and breast cancer, further expanding its potential applications in cancer diagnosis ,44,45. TPancreatic adenocarcinoma is often considered a metastatic disease, as most cases present with local and metastatic recurrences and only 10–15% of cases are surgically resectable ,46. WhilAlthough breast cancer is known as a rare disease in male patients, the authors suggest that its metastases should be considered in various peripheral sites, including regions previously unsuspected and those recognized to have rare occurrences ,39,40,41As demonstrated, a comprehensive investigation should be conducted when there are unusual findings to rule out any rare occurrences. Our review of the current literature yielded only five reported cases of patients with colon cancer with confirmed metastases to the prostate gland. The first was diagnosed in 1993, and the last was reported in 2013  9,10,1111,12,13.The “seed and soil hypothesis” underpins the understanding of metastatic dissemination to specific organs. In this conceptual framework, tumor cells, likened to “seeds”, release cytokines and proteins that interact with the microenvironment of distant sites referred to as the “soil”. Effective interaction between these cytokines and the microenvironment facilitates the homing of circulating tumor cells to distant locations, thus inducing metastasis. However, certain sites, such as the skin, spleen, and muscles, are less conducive to distant metastasis. Factors contributing to this may encompass reduced cardiac output, particularly for the skin; the presence of immune cells and immune surveillance in organs like the spleen; or an unsupportive environment due to factors such as low pH or anaerobic metabolism, as is characteristic of muscles.The atypical occurrence of metastasis at unusual sites can complicate the differentiation between a metastatic lesion and a second primary tumor. Radiologists and pathologists play a pivotal role in resolving this diagnostic ambiguity. Determining the distribution pattern of metastatic lesions, recognizing morphological similarities between sites of metastases, and, in more complex cases, conducting arterial and venous phase studies contribute to establishing the diagnosis of metastasis at unconventional locations . In instAs a general observation, encountering solitary rare metastases is infrequent; the overwhelming majority of rare metastatic occurrences tend to coincide with the emergence of metastases in other locations, underlying a progression towards more extensive systemic disease.In the realm of research, recent strides in comprehending the cellular and molecular intricacies of the metastatic process offer unparalleled potential for enhancing and formulating effective supplementary treatment approaches.On the clinical front, timely identification of secondary lesions remains the sole opportunity to manage the disease and extend survival. Consequently, familiarity with both common and uncommon sites of metastases empowers physicians to discern symptoms promptly and strategize optimal treatment plans.Of paramount importance is to consider that a better understanding of the metastatic process may reveal more therapeutic targets for future oncological treatment.Our reported article is the first to describe a male with metastatic breast cancer in the kidney. Moreover, we present two unique occurrences of metastases to the prostate that contribute to the growing body of literature describing less common metastatic findings. Finally, we describe rare metastases from pancreatic adenocarcinoma located in the heart and metastatic melanoma spreading to the pancreas. The follow-up of these patients can yield valuable information for the future treatment of other such cases with long odds of success.


In this study, a validated quality evaluation method with peony flower fingerprint chromatogram combined with simultaneous determination of sixteen bioactive constituents was established using UPLC-DAD-MS/MS. The results demonstrated that the method was stable, reliable, and accurate. The UPLC chemical fingerprints of 12 different varieties of peonies were established and comprehensively evaluated by similarity evaluation (SE), hierarchical cluster analysis (HCA), principal component analysis (PCA), and quantification analysis. The results of SE indicated that similar chemical components were present in these samples regardless of variety, but there were significant differences in the content of chemical components and material basis characteristics. The results of HCA and PCA showed that 12 varieties of samples were divided into two groups. Four flavonoids , five monoterpenes and their glycosides , three tannins , three phenolic acids , and one aromatic acid (8) were identified from sixteen common peaks by standards and liquid chromatography–mass spectrometry (LC–MS). The simultaneous quantification of six types of components was conducted with the 12 samples, it was found that the sum contents of analytes varied obviously for peony flower samples from different varieties. The content of flavonoids, tannins, and monoterpenes (≥19.34 mg/g) was the highest, accounting for more than 78.45% of the total compounds. The results showed that the flavonoids, tannins, and monoterpenes were considered to be the key indexes in the classification and quality assessment of peony flower. The UPLC-DAD-MS/MS method coupled with multiple compounds determination and fingerprint analysis can be effectively applied as a feature distinguishing method to evaluate the compounds in peony flower raw material for product quality assurance in the food, pharmaceutical, and cosmetic industries. Moreover, this study provides ideas for future research and the improvement of products by these industries. Paeonia suffruticosa Andr.) is a perennial deciduous shrub of the genus Paeonia in the Ranunculaceae family, known as the “King of Flowers”, and is distributed in most regions of China, especially in Luoyang, Heze, Tongling, Pengzhou, and other places where it is widely planted. In addition to being used as an ornamental plant, the root bark of peony is a famous traditional Chinese medicine (TCM), also known as “Dan bark”, which has the function of clearing heat and cooling blood, promoting blood circulation, and dispersing blood stasis [The peony . It can not only comprehensively and integrally reflect the types and quantities of chemical substances in traditional Chinese medicine, but also serves as the main basis for identifying and controlling the quality of medicinal materials, providing scientific guidance for the quality evaluation of medicinal materials. It combines chromatographic techniques, such as GC ,9 and LCPaeonia rockii from different origins and determined four components, namely, oxypaeoniflora, paeoniflorin, albiflorin, and quercetin, only including three monoterpenes and their glycosides, and one flavonoid compound. Moreover, only using the retention time of the reference substance to identify the chromatographic peak is prone to cause false-positive results, with low specificity and accuracy. LC–MS has been proven to be a powerful technique for the analysis and identification of natural compounds [At present, there are few reports on the basic research of chemical composition and quality control of peony flowers. In particular, there are few studies comparing the chemical composition characteristics of different varieties of peony flowers. HPLC methods have been developed for the QC of peony flower, but these methods have the limitations of long analysis times and providing limited chemical information . Yan et ompounds  and has ompounds ,22,23,24ompounds . In addiIn the present study, a rapid and reliable UPLC-DAD-MS/MS method of multiple components determination in combination with chromatographic fingerprint analysis was developed for quality evaluation of peony flowers. To our knowledge, this is the first report on the analysis of components in peony flowers using a UPLC-DAD-MS/MS technique. Four flavonoids, five monoterpenes and their glycosides, three tannins, three phenolic acids, and one aromatic acid were determined and chosen as standards for their bioactivities or high contents. A total of 22 compounds were detected and 16 components were quickly identified or tentatively characterized by comparing the UV data, accurate mass, and fragment information with the reference substance. Meanwhile, chemical fingerprints of 12 different varieties of peonies were established and comprehensively evaluated by SE, HCA, PCA, and quantification analysis. This provides a significant reference for the quality control of peony flowers and provides a research basis for further research on the functional activity value of different varieties of peony flowers.The chromatographic conditions were optimized to obtain the best separation, symmetric peak shapes, as well as shorter run time by varying the column, the mobile phase, gradient program, flow rate, column temperature, and detection wavelength.Columns such as Agilent Eclipse plus C18 column , Agilent prosell 120 EC C18 column , Agilent Eclipse SB C18 column , and Agilent prosell 120 SB C18 column  were examined and compared for their ability of separation resolution and retention, and the Agilent prosell 120 SB C18 column was confirmed to be the better choice. In particular, the peak effect of 10–12 min and 15–17 min was significant, as shown in The column temperatures  were also studied in this paper, and the results showed that with the increase in column temperature, the peak time was advanced, the separation degree of adjacent peaks was decreased, and the response value of analytes was increased. Under the condition that the sensitivity was satisfied, a good chromatographic separation could be achieved at 30 °C. During the selection of flow rate, it was found through comparison that with the increase in flow rate, the analysis time would be further shortened, but the resolution of the target components would become smaller and smaller, especially for components with similar polarity (such as Cn and Rn), which were particularly prone to overlap and could not achieve an ideal separation effect. Moreover, entering a higher flow rate in the mass spectrometry would reduce the ionization efficiency. In addition, due to the high flow rate, the drying airflow and pressure also need to increase, resulting in poor reproducibility and increased costs. Therefore, the desired separation and acceptable tailing factor can be obtained at the flow rate of 0.40 mL/min without building too much backpressure on the column. Moreover, a full wavelength scan (190–400 nm) was performed to select the best detection wavelength a, and foIn conclusion, the optimal separation was achieved on the Agilent prosell 120 SB C18 column at 30 °C with a flow rate of 0.40 mL/min, gradient elution system of acetonitrile with 0.1% aqueous formic acid, and wavelength at 270 nm (no reference), and the separation was accomplished within 40 min.To obtain satisfactory extraction efficiency, the extraction methods and extraction solvents were investigated. Compared with refluxing extraction, ultrasonic extraction was simpler and more economical and effective and did not cause the transformation of some unstable components due to the influence of temperature, and then used in further experiments. v/v/v) were investigated to obtain optimal extraction conditions by comparing the numbers, areas and resolution of the chromatographic peaks. The results showed that (v/v/v) at room temperature for 30 min was effective and convenient for the analytes’ extraction.For extraction solvents, aqueous methanol and ethanol solutions were tested. Firstly, in this study, four methanol aqueous solutions with different volume concentrations  were selected for extraction, and the results showed that  the extrwed that  with theAccording to the guideline of CFDA  and someThe injection precision was evaluated by successive analysis of the same sample solution six times. The results demonstrated that the RSDs of injection precision were in the range of 0.03–0.25% for the RRT and 0.14–1.55% for the RPA. The repeatability was evaluated with six independently prepared sample solutions. The RSDs of repeatability were in the range of 0.06–0.20% for the RRT and 1.04–4.04% for the RPA. The sample stability was evaluated by analyzing the same solution at different times . The RSDs of sample stability were below 0.20% for the RRT and 4.67% for the RPA. These results confirmed that the method of UPLC for the fingerprint analysis was valid and satisfactory ,26.The development and validated UPLC method with DAD detector was applied to investigate the fingerprint chromatograms of 12 PPP samples with different varieties. Load the DAD chromatogram of the test solution into the similarity evaluation software for the chromatographic fingerprint of traditional Chinese medicine (version 2012) . Data cuThe consequences of similarity evaluation were shown in The cluster analysis of 12 varieties PPP samples was performed by SPSS 26.0 soft-ware based on the similarities from 12 varieties of PPP samples. The input table of the HCA is listed in PCA was also performed on the RPA to ensure that all elements equally influenced the results ,29. ThroThe concentrations of the sixteen components, including Gd, Me, Oa, Pn, Ee, Aln, 3G, Bd, 4G, 5G, Asd, Cn, Rn, Mc, Bn, and Kl were quantitatively determined through UPLC-MS/MS. Under the selected chromatographic and mass spectrometric conditions, the typical ion flow chromatograms of the sixteen components in mixed standard samples and samples are shown in Y) versus concentrations  of identified constituents in the standard solutions. The regression equations, correlation coefficients (R), and concentration ranges are shown in The developed method was validated for its specificity, linearity, LODs and LOQs, injection precision, repeatability, sample stability, and accuracy. All calibration curves were plotted based on linear regression analysis of the integrated peak areas , ethyl gallate (Ee), methyl gallate (Me), paeoniflorin (Pn), albiflorin (Aln), apigenin-7-O-β-D-glucopyranoside , oxypaeoniflora (Oa), mudanpiosideC (Mc), benzoyloxypaeoniflorin (Bn), and rhoifolin (Rn)  was applied as follows: 0–8 min, 2–12% A; 8–11 min, 12–16% A; 11–15 min, 16–20% A; 15–20 min, 20–25% A; 20–24 min, 25–45% A; 24–30 min, 45–65% A; 30–33 min, 65–90% A; 33–36 min, 90–2% A; and 36–40 min, 2% A. DAD was set to scan from 190 to 400 nm, and 270 nm (no reference) was used as detection wavelength with the sample injection volume of 2 μL.UPLC analysis was performed on an Agilent1290 Infinity II system  equipped with a diode array detector (DAD), which was connected to Agilent Chemstation B.08 software. All separations were carried out on an Agilent prosell 120 SB C18 column  at a flow rate of 0.40 mL/min, maintained at 30 °C. A binary gradient elution system composed of acetonitrile (solvent A) and 0.1% aqueous formic acid (2) as collision gas after choosing the precursor ions and product ions of interest. ESI Mass spectra were acquired over the range from m/z 100 to 1000 in positive mode. Negative mode was also done when necessary to obtain further confirmation. Nitrogen was used as the drying gas at a flow rate of 11.0 L/min and the drying gas temperature was 350 °C; the nebulizing gas (N2) was set at 40 psi and the capillary voltage was set at 4500 V (ESI+) and 3500 V (ESI−), respectively. The data were analyzed using Mass Hunter Data Analysis B.07 software. The ion pairs, residence time, collision energy, and fragmentation voltage used for qualitative and quantitative analysis of 16 components detected by MRM method were listed in UPLC–ESI-QQQ-MS analysis was carried out on an Agilent1290 Infinity II system  in combination with a 6460 Triple Quad MS mass spectrometer which was equipped with an ESI source, using Agilent Mass Hunter workstation control B.08 software for data acquisition. The chromatographic conditions were as described above. The MS/MS spectra were obtained by CID with high-purity nitrogen . After taking them out, peony petal samples were comminuted with a mill to pass through 24-mesh sieve to obtain peony pollen samples and then put it in a sealed bag for standby.v/v/v) in a 100 mL volumetric flask. During the ultrasonic process, ice was put in to keep it at room temperature (25 °C) for ultrasonic extraction as much as possible. After cooling, the extracted solution was added to the original weight. A volume of 2 mL of the solution was filtered through a 0.22 μm PTFE filter before analysis.The accurately weighed powder (about 0.5 g) was extracted by ultrasonication for 30 min with 50 mL methanol-ethanol mixed aqueous solution . Recovery was investigated to check the accuracy of the method. Three different concentrations of mixed standard solutions were spiked into the same sample. Then, the samples were extracted and analyzed using the proposed method. Three replicates were performed for the test. Variations were expressed by RSD in all four tests above.Twelve varieties of peonies  were analyzed under the chromatographic condition of Similarity analysis was performed by professional software named Similarity Evaluation System for Chromatographic Fingerprint of TCM, which was recommended by China Food and Drug Administration. The correlation coefficients of entire chromatographic profiles of the samples were calculated, and the similarities of different chromatograms are based on the cosine ratio between sample chromatogram and reference chromatogram. Hierarchical clustering analysis (HCA) was performed by SPSS 26.0 . Ward’s method, which is very efficient for the analysis of variance between clusters, was applied, and squared Euclidean distance was selected as measurement for the analysis. For further discrimination among samples, principal component analysis (PCA) pattern recognition method was employed using Soft Independent Modeling of Class Analogy-P 11.5 Demo.In this study, a simple, accurate, and reliable UPLC-DAD-MS/MS method was developed and validated to evaluate the quality of PPP through fingerprint chromatogram combined with simultaneous determination of six types of components from sixteen analytes, namely flavonoids , monoterpenes and their glycosides , tannins , phenolic acids , and aromatic acids (8). Twelve samples were divided into two groups in HCA and PCA. In the quantitative determination, six types of components of the 12 varieties of PPP were successfully separated and determined. The content of flavonoids, tannins, and monoterpenes (≥19.34 mg/g) was the highest, accounting for more than 78.45% of the total compounds. The results demonstrated that the flavonoids, tannins, and monoterpenes were considered to be the key indexes in the classification and quality assessment of PPP. The UPLC fingerprint analysis is based on sixteen mainly bioactive constituents’ detection in different varieties of PPP samples combining SE, HCA, PCA, and quantification analysis can be effectively applied to evaluate the quality of the peony flower. The method proved to have good linearity, injection precision, repeatability, recovery, and sample stability. It is demonstrated that UPLC-DAD-MS/MS method coupled with multiple compounds determination and fingerprint analysis is a powerful, practical tool for comprehensive QC of peony flower. Thus, this study can be effectively used to evaluate the compounds in peony flower raw material for product quality assurance in the food, pharmaceutical, and cosmetic industries. Moreover, this study provides ideas for future research and the improvement of products by these industries.


ELN) and hemoglobin beta chain-beta S variant (HBB-BS), were found in all chambers, with 35 communal DEGs in the LA and LV and 15 communal DEGs in the LV and RV in both CH and HF. Functional enrichment analysis of these genes emphasized the crucial roles of the extracellular matrix and sarcolemma in CH and HF. Lastly, three groups of hub genes, including the lysyl oxidase (LOX) family, fibroblast growth factors (FGF) family, and NADH-ubiquinone oxidoreductase (NDUF) family, were determined to be essential genes of dynamic changes from CH to HF.Pressure overload-induced pathological cardiac hypertrophy (CH) is a complexed and adaptive remodeling of the heart, predominantly involving an increase in cardiomyocyte size and thickening of ventricular walls. Over time, these changes can lead to heart failure (HF). However, the individual and communal biological mechanisms of both processes remain poorly understood. This study aimed to identify key genes and signaling pathways associated with CH and HF following transverse aortic constriction (TAC) at four weeks and six weeks, respectively, and to investigate potential underlying molecular mechanisms in this dynamic transition from CH to HF at the whole cardiac transcriptome level. Initially, a total of 363, 482, and 264 differentially expressed genes (DEGs) for CH, and 317, 305, and 416 DEGs for HF were identified in the left atrium (LA), left ventricle (LV), and right ventricle (RV), respectively. These identified DEGs could serve as biomarkers for the two conditions in different heart chambers. Additionally, two communal DEGs, elastin ( Cardiovascular diseases are the leading cause of death worldwide and are associated with immense health and emotional and economic burdens . Heart fIn this study, we developed a pressure overload mouse model using the transverse aortic constriction (TAC) strategy to systematically analyze and elaborate the gene expression patterns in different chambers of the whole heart. By analyzing the transcriptomic data of samples from different compartments, we can reveal more molecular markers involved in the two types of disease and identify the critical factors responsible for the transition from CH (four weeks post-TAC) to HF (six weeks post-TAC). Our results will help to shed new light on the pathogenesis of CH and HF and provide additional material and a research base for subsequent studies on CH and HF.n ═ 27) were anesthetized by intraperitoneal administration of 1% pentobarbital (0.005 mL/g body weight). TAC surgery was performed as previously described [n ═ 3 per group).Male C57BL/6J mice . We collected 27 RNA samples of all conditions from nine mice, including three CH mice, three HF mice, and three Sham mice. RNA purity, concentration, and integrity were checked and measured using the Bioanalyzer 2100 system . Sequencing libraries were prepared from 3 µg of high-quality RNA per sample according to the manufacturer’s recommendations. After clustering of the index-coded samples, library preparations were sequenced on an Illumina Hiseq platform and 150 bp paired-end reads were generated.https://www.ncbi.nlm.nih.gov/assembly/GCF/_000001635.20/). We aligned the clean RNA sequencing (RNA-seq) data to the GRCm38 reference genome using Hisat2 v2.0.5 [http://broadinstitute.github.io/picard) to assign read group information which includes library, lane, and sample identity and to remove duplication caused by PCR.We filtered raw data in “fastq” format using in-house Perl scripts and Trimmomatic v. 0.35 software . After r2 v2.0.5 . SubsequIn our analyses, a gene or isoform was considered ubiquitously expressed if its Fragments Per Kilobase of transcript sequence per Millions base pairs (FPKM) was greater than 0.1 across all samples. To identify DEGs between different conditions, the value of one was added to each FPKM value before the log2 transformation. Gene expression levels in each sample were quantified as read counts generated with featureCounts v1.5.0-p3 . Subsequ2R (Pearson correlation coefficient) between biological replicate samples to be at least greater than 0.7. A sample correlation heat map was drawn according to the correlation coefficient between samples. PCA analysis was performed for FPKM using the SMARTPCA program in the EIGENSOFT package v. 5.0.2 [Correlation coefficients were calculated based on the FPKM values of all genes in each sample to visually represent sample differences between groups and duplication within groups. In this study, we required v. 5.0.2 , and eigP values were adjusted using Benjamini and Hochberg’s approach to control the false discovery rate. Genes with an absolute value of log2FC greater than one and an adjusted P value less than 0.05 as determined by DESeq2 were assigned as differentially expressed. After normalizing the rows of expression data, we used hierarchical clustering to perform cluster analysis on the gene expression values of the DEGs. We created a heat map of clustering and a Venn diagram using R language. Through the Venn diagram, it is possible to identify communal or individual differential genes for a particular comparison combination.Differential expression analysis between any two comparisons was performed using the DESeq2 R package v. 1.16.1 . The resP value less than 0.05 was considered significantly enriched by DEGs. To further investigate various reactions and biological pathways, we conducted a Reactome analysis using the “ReactomePA” package in R. An adjusted P value less than 0.05 was used as the threshold of significant enrichment in the Reactome pathway [To analyze the screened DEGs at the functional level, we performed Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the “clusterProfiler” package in R language . A corre pathway .http://string-db.org/), which predicts PPIs. An interaction with a combined score greater than 0.4 was considered statistically significant. Subsequently, the PPI networks were visualized using Cytoscape v.3.6.1 [PPI analysis of DEGs was conducted using the STRING database  and adhered to the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.GraphPad Prism 7  and presented as mean ± standard deviation (SD). The Shapiro–Wilk test was used for the normal distribution assay and Pearson correlation was utilized for correlation analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey’s method or unpaired two-tailed Student’s t-tests. A P value less than 0.05 was considered to indicate a significant difference. The Benjamini–Hochberg procedure was used to control the false discovery rate.All data were analyzed using R statistical software  and We performed 2-D-guided M-mode echocardiography on mice four and six weeks post-TAC and Sham surgery . LVIDd wTable S1. Q20 and Q30 of all clean reads were more than 99.2% and 95.8%, respectively, indicating that the RNA-seq data were of high quality for transcriptome analysis.To construct a comprehensive transcriptomic heart map of the mouse following TAC, we generated an RNA-seq data set using 27 samples isolated from eight weeks old C57BL/6J male mice. Approximately 127.2 million paired-end raw reads were generated on average, yielding 125.5 billion clean reads and 188.3 G base pairs for the study after quality control. Further details of the RNA-seq data are provided in Figure S1A. After removing zero values, adding one and applying a log2 transformation to the FPKM values, a final data matrix of 34,793 genes was generated for subsequent analyses. The distribution of gene expression levels across different samples using the final FPKM matrix is illustrated in Figure S1C. Both correlation analysis and PCA were performed on all models based on their FPKM values. The results showed that Pearson correlation coefficients (2R) were all greater than 0.7 in the correlation analysis, and samples were clustered according to their source in the PCA analysis, as seen in Figure S1B. These results demonstrated that three different heart chambers exhibited unique gene-expression profiles.With all the biological samples combined, a raw data matrix of FPKM values was generated. This consisted of 54,533 genes annotated in the Ensembl GRCm38 mouse reference genome across 27 samples. Most of the reads were in genic regions as shown in P value less than 0.05 and the absolute value of log2FC greater than one. Similarly, in the LV, 482 DEGs, including 332 up-regulated and 150 down-regulated DEGs, and in the RV 264 DEGs, including 165 up-regulated and 99 down-regulated DEGs, were identified, respectively . These genes are closely related to cardiac remodeling, cardiac rhythm, cardiac contraction, energy, and oxygen delivery, and several HF marker genes were detected, suggesting these genes play an essential role in HF. The top 10 up-/down-regulated DEGs  may serve as potential biomarkers of CH and HF in different heart chambers. The top 10 DEGs for each chamber in CH and HF are also listed in The DESeq2 software was used to identify DEGs in the CH and HF in left atrium (LA), LV, and right ventricle (RV), respectively, compared to Sham corresponding to the same regions of heart. For the CH group, 363 DEGs, including 68 up-regulated and 295 down-regulated DEGs, were identified in the LA using criteria of ectively . The mosFigure S2). Moreover, GO enrichment of DEGs can aid in further classification and enhance understanding of their functions in different diseases. For CH, the most significant GO terms associated with DEGs in the LA and LV were biological processes and cellular components except in the case of the RV. Three GO terms related to extracellular matrix (ECM), structure, and encapsulating organization were shared across all three chambers. In contrast, muscle contraction, muscle system process, and collagen-containing ECM were shared in LA and LV. Other GO terms were specific to each chamber: GO terms related to smooth muscle contraction were identified in the LA, while GO terms associated with cardiac muscle cell membrane repolarization, myosin filament, and sodium channel complex were found in the LV. GO terms linked to energy metabolism and transmembrane transporter activity of a substance were observed in the RV . However, for the HF group, the top enriched GO items in the LA were mainly associated with circadian rhythm, substances involved in reduction–oxidation reaction (REDOX) processes, nerve conduction, and oxygen/gas transport. Apart from the five GO terms related to ECM structure and composition shared in the LV and RV, the most significant GO terms in the LV were related to amino acid transmembrane transport, regulation of cardiac/striated muscle cell apoptotic process, fat cell differentiation, and chemokine/cytokine/immune receptor activity. Finally, GO terms in the RV were associated with muscle/myotube/striated muscle cell differentiation, myocardial structure development and maintenance, including sarcomere, myofibrils, and contractile fibers . Detailed information on the GO terms in CH and HF from the three individual chambers are shown in Table S2.To explore the biological processes, cellular components, and molecular functions associated with DEGs, we performed GO analysis for the DEGs in the two different pathological conditions . The detailed KEGG and Reactome pathways of CH and HF in the LA, LV, and RV can be found in Tables S3 and S4, respectively.To comprehensively investigate biological systems and metabolic pathways, we conducted KEGG and Reactome pathway enrichment analyses for the DEGs in the two conditions. While Reactome emphasizes comparative analysis of biological reactions and physiological processes, KEGG primarily addresses gene function and metabolic pathways. For CH, the most significant KEGG and Reactome pathways in the LA, LV, and RV are shown in Figure S2 and S3 display the similarities and differences in gene expression patterns among the various chambers in CH and HF, respectively. However, there were significant differences in the numbers of DEGs among the LA, LV, and RV in the two different conditions  and hemoglobin beta chain-beta S variant (HBB-BS), were shared across the whole heart, suggesting a change in mechanisms during the development of HF after TAC. Elastin is an ECM protein with a unique biochemical structure that provides elasticity to organs and tissues, including the heart and blood vessels. Both genetic and acquired cardiovascular diseases are associated with elastin and elastic fibers, which inhibit smooth muscle cell proliferation and promote actin filament bundles’ organization. Elastin-derived peptides, or elastokines, stimulate the migration and proliferation of monocytes and fibroblasts [HBB-BS gene, encodes a beta polypeptide chain found in adult hemoglobin, which transports oxygen to various peripheral tissues. It is associated with glucose/energy metabolism, suggesting changes of oxygen delivery and energy in CH and HF. These results preliminarily indicate that ELN and HBB-BSS could be potential clinical biomarkers or therapeutic targets for CH and HF. However, further studies are urgently needed to confirm these findings. In addition, we performed a separate analysis of the expression of nine potential indicator genes associated with cardiac structure/function and fibrosis, including ELN, HBB-BS, MYH6, MYH7, NPPA, NPPB, COL1A1, COL1A2, and COL3A1. Their expressions were significantly higher at four and six weeks compared to the Sham group, suggesting that CH and remodeling had already occurred by the fourth week . The fibrosis-related genes COL1A1, COL1A2, and COL3A1 showed even higher expression in the CH group than in the HF group in LA and LV. These results are associated with extensive ECM remodeling in the LA and LV, suggesting that the fibrosis-related genes may dynamically change in the mediation of HF . Heat maps were constructed using the genes reported to be associated with CH and HF in our dataset . The results showed that the expression of these genes exhibited distinctive changes in the LA, LV, and RV.In this study, we conducted a comparison between CH and HF at the whole heart level, taking into account the different chambers. This approach addresses the integrative physiological effects of TAC at both the CH and HF stage. We outlined the transcriptome profiles of the whole heart under CH and HF conditions. Compared with the Sham group, we identified hundreds of unique DEGs in each of the three chambers under these two pathological conditions. Only two DEGs, elastin , 102 (LV), and 85 (RV) common DEGs between the CH vs Sham and HF vs Sham at entire heart levels, which indicates that these shared DEGs play a crucial role in the pathogenesis of HF in the whole heart and exhibit similar damage effects in individual chambers. Although the top 10 DEGs in the two different states were rarely identical across different compartments, most of their GO items were similar, mainly related to the ECM, muscle system processes/contraction, and cardiac conduction. Notably, fat cell differentiation, muscle cell differentiation/development, apoptosis of striated muscle cells, and cytokine binding were specific to HF, indicating that the structural remodeling of the heart is more intensified, and the inflammation is more severe than in the CH.In the LA, the pyruvate, butanoate, drug, xenobiotics metabolism, and fatty acid degradation pathways were specific to CH. This indicates a shift in atrial metabolism and an accumulation of metabolic small molecule products in CH, but not in HF.In the LV, which is the primary area affected by TAC, several pathways are shared between CH and HF. These include chemokine, cytokine, adrenergic, aldosterone related, platelet activation KEGG pathways, as well as ECM, collagens, laminin, and integrin-related Reactome pathways. The results suggest that a greater number of inflammatory pathways are involved in the occurrence and progression from CH to HF, and that cardiac structure and conduction pathways are altered. Moreover, the involvement of ECM and collagen fiber-related pathways in both states suggests that fibrosis is developing in the myocardium of the LV, even though myocardial hypertrophy may serve as a compensatory mechanism to maintain the integrity of the function and structure of the damaged heart. Fibrosis occurs due to the excessive deposition of many ECM proteins in myocardial tissue caused by the long term increase of pressure load. This pathological remodeling process of myocardial interstitial expansion will eventually increase myocardial stiffness, resulting in final HF. Notably, activation of both the sympathetic nervous system and the renin-angiotensin–aldosterone system is sufficient to cause cardiomyocyte death . HoweverPKG activity increases the resting tension in myocardial cells, leading to myocardial hypertrophy, fibrosis, and vascular stiffness in patients with HF [In the RV, the KEGG pathways, such as the MAPK signaling pathway and adrenergic signaling in cardiomyocytes, continue to play crucial roles in CH. Furthermore, an increased number of immune cell-related KEGG pathways were found to be enriched in CH. The cellular components of the immune response, including T cells, B cells, mast cells, natural killer cells, and dendritic cells, are associated with the pathogenesis of CH. Concurrently, the energy metabolism and muscle contraction Reactome pathways suggest that an enhanced myocardial energy metabolism facilitates muscle compensation, maintaining normal function during CH. Contrarily, apart from ECM and muscle contraction, the hormone pathways, insulin secretion, TNF signaling pathway, and O-linked glycosylation were significantly enriched in HF. It is known that overexpression of TNF-α in normal myocardium directly causes myocardial insulin resistance, aggravating cardiac dysfunction and ventricular dilatation following ischemia. Low  with HF , 32. In  with HF .LOXL2, LOXL3, FBN1, MFAP5, and MFAP2 as hub nodes with the most incredible degrees in the MCODE sub-network which had the highest score for the shared 2 DEGs. Similarly, MAPT, NDUFB2, NDUFAB1, NDUFS5, and NDUFB4 were identified in the LV and RV, while FGFR1, FRS2, FGF10, FGF2, and MMP2 were identified in the LA and LV. Lysyl oxidase (LOX) family-associated genes, including LOX and LOXL1, and the ELN gene were significantly enriched in the sub-network modules of the three comparison groups. As mentioned earlier, when the integrity of the elastic fiber is compromised, ECM proteins are produced and remodeled, which can further alter the mechanical behavior of the cardiovascular system [Fibrillin 1 (FBN1) gene encodes a member of the fibrillin family, the encoded protein being a large ECM glycoprotein and a structural component of 10–12 nm calcium-binding microfibrils. MFAP2 and MFAP5 are major antigens of elastin-associated microfibrils and are candidates for involvement in ECM organization and elastic fiber formation.To identify the key genes and their bio-signature in the progression from CH to HF at the LV, LA, and RV, we first screened 35 overlapping DEGs in two different states in the LA and LV and 15 common DEGs in the LV and RV. Functional analysis revealed that the 35 shared and 15 common DEGs are mainly involved in the ECM, cardiac structure or function, and signal transduction. Following this, we identified the top five genes, including r system . It is bMMP2 has been identified as a potential biomarker of myocardial fibrosis in patients with hypertrophic cardiomyopathy [NDUF) gene family transfers electrons from NADH to the respiratory chain. In summary, the critical hub genes we screened have high confidence.In cardiovascular disease, the fibroblast growth factors (FGFs) family is implicated in cardiac remodeling through different mechanisms, which can contribute to HF. FGF2 induces CH by activating FGF receptor 1c (FGFR1c) and MAPK signaling. FGF receptor substrate 2α (FRS2α) is the primary mediator of signaling in the FGF pathway. Multiple epidemiological and patient studies have concluded that FGF23 acts directly on the myocardium or via elevations in blood pressure to cause left ventricular hypertrophy . The hormyopathy . NDUFB2,This study is based on a relatively small sample size which warrants discussion. Although our data have demonstrated that CH occurred four weeks post-TAC, and a significant decline in cardiac function and HF occurred at six weeks post-TAC. When measuring cardiac function using echocardiography, it is advisable to include more parameters to obtain comprehensive indicators of both long-axis and short-axis, such as interventricular septal diameter at end-diastole (IVSd) and left anterior descending artery diameter at end-diastole (LADd) as well as heart weight and lung weight measurements. In addition, regarding the expression of hub genes in dynamic changes of CH and HF, this article presents a bioinformatics analysis study that still requires experimental and external verification. Our future work will involve experimental validation of these results and focus on exploring the biological functions and molecular mechanisms.ELN and HBB-BS might be potential biomarkers in patients with CH and early HF based on the comprehensive analysis conducted across three cardiac chambers. Furthermore, we demonstrated that gene expression during the development of HF is characterized by dynamic sets of overlapping transcripts regulated by specific interrelated mechanisms. Notably, each cardiac chamber exhibits unique gene expression patterns and regulatory mechanisms. Our results showed the cardiac ECM is composed of numerous molecules, including collagen, elastin, fibrin, lox, MMPs, fibronectin, laminin, fibrillin, glycoproteins, and proteoglycans. The ECM is forming a dynamic environment that plays an active and crucial role in regulating cellular events and cardiac remodeling [FGFs family, and respiratory electron transport-associated genes play significant roles in the dynamic progression of HF. These genes along with their associated signaling pathways contribute to the identification of independent DEGs, providing new insights into the distinct mechanisms underlying CH and HF. The common DEGs identified in this study shed light on the potential shared mechanism of CH and HF. Furthermore, the identified hub genes have the potential to serve as biomarkers or therapeutic targets in hypertrophy and early HF.In conclusion, our findings suggest that modeling , 39. ECMhttps://www.bjbms.org/ojs/index.php/bjbms/article/view/8997/2815.Supplementary data are available at the following link:


Three years ago, our patient, at that time a 16-month-old boy, was discovered to have bilateral kidney lesions with a giant tumor in the right kidney. Chemotherapy and bilateral nephron-sparing surgery (NSS) for Wilms tumor with nephroblastomatosis was carried out. The patient also had eye affection, including glaucoma, eye enlargement, megalocornea, severe corneal swelling and opacity, complete aniridia, and nystagmus. The diagnosis of WAGR syndrome was suspected. De novo complex chromosomal rearrangement with balanced translocation t and a pericentric inversion inv(11)(p13q12), accompanied by two adjacent 11p14.1p13 and 11p13p12 deletions, were identified. Deletions are raised through the complex molecular mechanism of two subsequent rearrangements affecting chromosomes 11 and 10. WAGR syndrome diagnosis was clinically and molecularly confirmed, highlighting the necessity of comprehensive genetic testing in patients with congenital aniridia and/or WAGR syndrome. PAX6 and WT1 genes, serve as a cause of the syndrome [WAGR syndrome OMIM #194070) is a rare chromosome syndrome that occurs in 1 child per 1,000,000 births 94070 is . Additiosyndrome . ComplexHere we report an extremely rare case of a patient with a giant early developed embryonic renal tumor, glaucoma, keratopathy, and complete bilateral aniridia associated with a complex chromosomal rearrangement, balanced translocation t, pericentric inversion inv(11)(p13q12), and two adjacent deletions at the breakpoints of the complex chromosomal rearrangement.A 3100 g, 48 cm Caucasian boy was born to a 29-year-old woman at 38 weeks of gestation. He was born with a loop of cord and fetal hypoxia in labor. Apgar scores were 8 and 9 at the 1 and 5 min. The patient was born in a second pregnancy in a non-consanguineous family. His parents are healthy. The infant was discharged with the diagnoses of congenital bilateral complete aniridia, hypospadias, cryptorchidism, and mild hypotonia.At the age of 12 months, the patient referred to the ophthalmologist with complaints of corneal opacity, eye enlargement, and nystagmus. An ophthalmologic examination included corneal biomicroscopy, gonioscopy, intraocular pressure measurement, and retina examination. A fine horizontal nystagmus was noted. Corneal biomicroscopy revealed initial corneal pannus in the shape of a thin strip without vascularity, megalocornea, cornea swelling, complete aniridia, and glaucoma. The central corneal thickness was 720 µm and 664 µm in R/E and L/E, respectively . The retina examination revealed disc pallor, normal cup, and absence of the foveal reflex. Gonioscopy confirmed hypoplasia of the iris and mesodermal tissue rests in the anterior chamber angle. The intraocular pressure (IOP) measured with rebound tonometry  was 27 mm Hg and 29 mm Hg in the R/E and L/E, respectively . Since 12 months of age, the patient received initial treatment of topical anti-glaucoma medication with positive dynamics.At the age of 13 months, a control ophthalmologist examination showed a positive effect of conservative therapy. The proband was visually rehabilitated with photochromatic glasses. Preservative-free artificial tears eye drops (carboxymethylcellulose) were prescribed for aniridia-associated keratopathy (AAK).At the age of 14 months old, the patient, with vomiting, diarrhea, and subfebrile temperature, was referred local hospital. Clinical and radiological examination revealed a big right kidney tumor (Wilms tumor) with left kidney lesions suspicious of nephroblastomatosis. The patient was transferred to the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology for diagnosis and medical care.Chemotherapy, according to the SIOP-RTSG 2016 Umbrella protocol, was started, a regimen for bilateral disease with predisposition syndrome. The patient underwent 6 weeks of AV without a good response. An MRI showed a 20% increase in right kidney tumor size. The therapy scheme was switched and intensified with one course of VP16/Carbo, but with no good response either; on the 11th day of the course, the abdominal circumference increased to 55 cm. According to the CT, there was a 40% increase in the right kidney tumor, and left kidney lesions seemed to be without dynamics. The lack of response to chemotherapy might have indicated a histological stromal type of Wilms tumor. Thus, a decision was made to perform surgery instead of chemotherapy prolongation. Transverse laparotomy and both kidneys NSS were performed. Histology showed WT, stromal type, intermediate risk, local stage II  in the right kidney, and nephroblastomatosis lesions in the left one. Adjuvant chemotherapy, in this case, was performed according to the AV-2 scheme (27 weeks), and maintained chemotherapy for nephroblastomatosis was given monthly up to one year from the start of neoadjuvant treatment.The child achieved a full response and was left under observation with a follow-up duration of 37 months from the start of the treatment.Because of a congenital penis defect at the age of 18 months, orthoplasty of the penis, Bracka two-stage repair surgery, was carried out. Functional gonadal tissue was defined. The patient suffers from secondary obstructive pyelonephritis, frequent relapses, incomplete doubling of the right ureter, and secondary obstructive ureterohydronephrosis of the doubled segment of the right kidney.At the age of three years, the patient’s ocular manifestations included congenital complete aniridia, nystagmus, fovea hypoplasia, drug-compensated glaucoma, aniridia-associated keratopathy (stage 1), ptosis (degree 1), and high myopia .The patient has a mild delay in speech and motor development and no other neurological disorders.At the age of 2 years old, the patient was referred to the Research Centre for Medical Genetics, where WAGR syndrome was diagnosed. In order to confirm the diagnosis, the patient underwent a series of genetic testing.During a standard cytogenetic analysis, abnormal male karyotype 46,XY, accompanied by complex rearrangement, specifically reciprocal translocation between short arms of chromosomes 10 and 11 t, was revealed. A pericentric inversion inv(11)(p13q12) was also suspected but required additional investigation . DerivatRegion-specific FISH analysis for the short and long arms of chromosome 11 demonstrated that a fragment of the short arm of derivative chromosome 11 appeared to be situated under the centromere on the long arm of this derivative der(11) chromosome. These findings confirm the additional pericentric inversion of derivative chromosome 11, inv(11)(p13q12). Additional mBAND confirmed the results of the FISH analysis . The anaWT1 (MIM #607102) and PAX6 (MIM #607108) genes, which correlated with the clinical phenotype of WAGR syndrome in the patient.Furthermore, to determine the possible presence of cryptic imbalances that occurred during chromosome rearrangements, chromosomal microarray analysis (CMA) was performed . The anaThe patient with complex chromosomal rearrangements and local imbalance at the breakpoints of chromosome 11p13 presented all four classical features of WAGR syndrome: early developed bilateral renal tumor, congenital aniridia, genitourinary anomalies, and intellectual disability. He was born with a birth weight of 4.8 kg (median is 2.9 kg), and he was diagnosed with Wilms tumor at the age of 15 months, which is very early since the median age of WAGR-associated Wilms tumor diagnosis is 22 months [In the neonatal period and early infancy, patients with isolated aniridia and with WAGR syndrome are clinically undistinguishable . Indeed,Nevertheless, the patient described here was first referred to the oncologist for tumor treatment. In this case, nephroblastoma developed very early. Regular oncological surveillance was conducted, which showed complete remission with no sign of relapse, with secondary obstructive hydronephrosis on the right.In the patient, congenital aniridia combined with nystagmus, glaucoma, optic nerve hypoplasia, and fovea hypoplasia are the most common features of the disease ,8. OculaUnder the supervision of various specialists, neurologists, psychologists, and special teachers, the boy, who is now 4 years old, attends a pre-school with a special educational program. We may conclude that the patient had a relatively mild course of the disease except for the early development of Wilms tumor, which nevertheless was successfully treated.PAX6 and WT1 gene was derived due to the reciprocal translocation between the short arms of chromosomes 10 and 11. Nevertheless, according to our data, this does not always lead to the development of WARG syndrome [LMO2 gene is affected by deletion. The first upstream deletion in the patient does not affect the LMO2 gene, so we could hypothesize his chances of developing a Wilms tumor with only that deletion were low. Nevertheless, according to the proposed molecular mechanism, a second rearrangement happened, specifically pericentric inversion in derivative chromosome 11, inv(11)(p13q12), resulting in a second downstream contiguous deletion 11p13p12 located adjacent to the first one. This deletion disrupted the LMO2 gene and was associated with a significant increase in the risk for Wilms tumor development [It was established that chromosome 11p13 imbalance occurred two times. It is interesting that the only imbalance that occurs during this complex chromosomal rearrangement is deletions on the 11p region. Chromosome 11 is known as a hot spot for breakage in chromosomal rearrangements . The firPAX6 haploinsufficiency. It is a key regulator of eye development. It is required to initiate developmental pathways, execute specification and differentiation programs, integrate information, synchronize processes in different eye tissues, and interact with other regulatory pathways [PAX6 also plays an important role in brain development [PAX6 function deficit due to the deletion could contribute to WAGR patients’ phenotype in several ways. The deficit leads to eye affections either in the shape of classical aniridia complex or aniridia-like phenotype when iris hypoplasia and other eye anterior segment abnormalities combine with foveal hypoplasia. PAX6 function deficit could also contribute to the neurological phenotype of WAGR patients and their brain integrity [A sufficient role in the patient’s phenotype is played by pathways . PAX6 alelopment . PAX6 funtegrity ,15.The diagnosis of congenital aniridia was based on the clinical presentation, including absence of the iris, foveal hypoplasia, and nystagmus. The severity of AAK was staged as defined by Holland et al. . Stage ICytogenetic analysis of the patient and her parents was performed on GTG-banded metaphase spreads obtained from cultured peripheral blood lymphocytes according to standard procedures. GTG-banded metaphase chromosomes were analyzed using Axio Imager A1 microscope  with Ikaros Karyotyping System Software, V.5.8.14 . The results were presented according to the International System for Human Cytogenomic Nomenclature 2020 .FISH was carried out using chromosomal preparations from cultured peripheral blood lymphocytes following the manufacturers’ protocols. DNA probes were used for subtelomeric regions of the short arm of chromosome 10 and the short arm of chromosome 11 ; whole-chromosome probe was used for chromosome 10, the short arm, and the long arm of chromosome 11 ; and Satellite Enumeration Probe was used for chromosome 11 (CEP 11-SE 11 (D11Z1), KREATECH, Amsterdam, The Netherlands). Multicolor Banding DNA probe on chromosome 11  was applied. FISH results were analyzed using an AxioImager M.1 epifluorescence microscope  and Isis digital image processing software . DNA probes for the chromosome 11-WT1/CEN11p  were applied. FISH results were analyzed using an AxioImager M.1 epifluorescence microscope  and Isis digital image processing software .http://dgv.tcag.ca/dgv/app/home, accessed on 9 September 2021), DECIPHER , and OMIM . Genomic positions refer to the Human Genome February 2009 assembly (GRCh37/hg19). The pathogenicity of variants was evaluated according to the American College of Medical Genetics (ACMG) standard guidelines [The CytoScan HD array  containing 2.67 million markers using the GeneChip™ 3000 system  was applied to detect the CNV across the entire genome, following the manufacturer’s protocols. Microarray-based copy number analysis was performed using the Chromosome Analysis Suite software version 4.0 , and the results were presented on the International System for Human Cytogenomic Nomenclature 2020 . Detected CNVs were totally assessed by comparing them with published literature and the public databases: Database of Genomic Variants (DGV) (idelines . DNA samThe clinical case highlights the need for numerous further studies to develop a common strategy for diagnosis and treatment of this nosology. Wilms tumor should be rapidly revealed in patients with aniridia as a symptom to prevent life-threatening complications. Our study also emphasizes the actuality of cytogenomic approaches in a postgenomic era.


While progress has been made in surveying the oceans to understand microbial and viral communities, the coastal ocean and, specifically, estuarine waters, where the effects of anthropogenic activity are greatest, remain partially understudied. The coastal waters of Northern Patagonia are of interest since this region experiences high-density salmon farming as well as other disturbances such as maritime transport of humans and cargo. Here, we hypothesized that viral and microbial communities from the Comau Fjord would be distinct from those collected in global surveys yet would have the distinctive features of microbes from coastal and temperate regions. We further hypothesized that microbial communities will be functionally enriched in antibiotic resistance genes (ARGs) in general and in those related to salmon farming in particular. Here, the analysis of metagenomes and viromes obtained for three surface water sites showed that the structure of the microbial communities was distinct in comparison to global surveys such as the Tara Ocean, though their composition converges with that of cosmopolitan marine microbes belonging to Proteobacteria, Bacteroidetes, and Actinobacteria. Similarly, viral communities were also divergent in structure and composition but matched known viral members from North America and the southern oceans. Microbial communities were functionally enriched in ARGs dominated by beta-lactams and tetracyclines, bacitracin, and the group macrolide–lincosamide–streptogramin (MLS) but were not different from other communities from the South Atlantic, South Pacific, and Southern Oceans. Similarly, viral communities were characterized by exhibiting protein clusters similar to those described globally (Tara Oceans Virome); however, Comau Fjord viromes displayed up to 50% uniqueness in their protein content. Altogether, our results indicate that microbial and viral communities from the Comau Fjord are a reservoir of untapped diversity and that, given the increasing anthropogenic impacts in the region, they warrant further study, specifically regarding resilience and resistance against antimicrobials and hydrocarbons. Coastal and estuarine environments are a hotspot for disturbances of anthropogenic origin ,2,3. ResThere is a global interest in monitoring ARGs in the natural and built environment ,16,17. RWhile ARGs are naturally occurring and ancient, e.g., presence of vancomycin resistance genes in 30,000-year-old permafrost in the Yukon, humans have contributed to their current ubiquity and prevalence ,27. The On the other hand, in coastal and estuarine environments in Patagonia and other relatively pristine environments, microbial and viral biodiversity is poorly understood, especially in comparison to global open ocean surveys. Expeditions such as Tara Oceans, Malaspina, the Global Ocean Sampling, and GEOTRACES have generated terabytes of shotgun metagenomic information, uncovering immense microbial and viral diversity as well as environmental drivers, metabolic strategies, and functional diversity ,38,39,40Patagonia, i.e., the region of southern South America from 40° S and higher, is traditionally regarded as pristine, even though industries such as agriculture (livestock) and salmon farming are commonplace. Similarly, due to the fragmented nature of the geography, land access to Patagonia is hampered by fjords and channels, especially west of the Andes Mountains. This results in commercial and passenger transport conducted mainly by sea routes on cruise ships, ferries, and small boats, all of which use hydrocarbons as a power source.Northern Patagonia, including the Comau Fjord, is impacted by high-density salmon farming, which results in the export of particulate organic matter to the sediments and an increase in dissolved inorganic nitrogen and dissolved organic nitrogen, all of which have been associated with harmful algal blooms. Additionally, the salmon farming industry poured more than 370 tons of antibiotics into the ocean in 2020, which is a hazard to local biodiversity and possibly to human health .In this study, we hypothesized that the Comau Fjord viral and microbial communities will be distinct from those collected in global surveys, but they will bear the hallmarks of microbes from coastal and temperate regions. Particularly to this study, we also hypothesized that microbial communities will be functionally enriched in ARGs in general and in those related to salmon farming in particular. For this, we sampled three surface coastal sites in the Comau Fjord and obtained metagenomes and viromes to test whether this under-sampled part of the world offers any taxonomic and functional diversity as compared to a global survey of the open ocean, in particular those publicly available from the Tara Oceans expedition. We also compared our results against global databases such as IMG, NCBI’s RefSeq, and GOS to obtain taxonomic and biogeographical information and functionally analyzed the metagenomes regarding functions and their resistomes, as well as viral communities, which showed a certain degree of uniqueness in their protein content.We collected 40 L of water at 5 m below sea level at 3 sampling sites  at the head of the Comau fjord located in Northern Patagonia (42° S) using an electric water pump . The fjord is characterized by seasonal variability associated with freshwater inputs from nearby rivers , a mean depth of 250 m (a maximum of ~480 m), 41 km of length and 4.5 km of width, precipitation (>5000 mm), and ice thawing in the summer season. During water collection in February 2016, we used a 50 μm mesh to avoid macroscopic debris. Water samples were immediately stored at 4 °C for no more than 48 h until processing. Then, we assembled a filtration system using a peristaltic pump to run the 40 L of water from each sample through a 0.22 μm Sterivex filter to capture prokaryotic organisms and subsequently through a 100 kDa tangential concentrator to concentrate viral particles. These resulted in prokaryotic and viral fractions for each of the 3 water samples, named Bac S1/S2/S3 and Vir S1/S2/S3, respectively. TM System . DNA was quantified in a Qubit fluorometer and used for the preparation of paired-end libraries according to the Illumina sample preparation guide. Libraries were subsequently sequenced on an Illumina MiSeq instrument at the Plant Biotechnology Center, Universidad Andrés Bello . Subsequently, we evaluated the quality of the 250 bp long paired-end reads using FastQC v0.11.8 and performed the filtering and trimming of the raw data using PRINSEQ v.0.20.4  [We used phenol-chloroform to isolate DNA from both prokaryotic and viral fractions. In addition, prokaryotic DNA was obtained by Xanthogenate nucleic acid isolation, as described in Alarcón-Schumacher et al., 2019. Viral DNA was extracted from viral particles purified by a CsCl gradient as described in Guajardo-Leiva et al., 2021 ,44. DNA ld > 20) ,46. We uSeawater was collected by pumping through a 50 μm net to exclude large organisms and stored in dark pet drums until arrival at the laboratory. Two liters of seawater were filtered through two 0.7 μm precombusted GF/F filters  and stored in the dark at −80 °C until chlorophyll-a (Chl-a) extraction and organic carbon and nitrogen determination (POC and PON). Additionally, 15 mL (in triplicates) of seawater was filtered through 0.2 µm mixed cellulose filters (Sartorious) and collected in polyethylene bottles for salinity, nitrate, nitrite, phosphate, and silicic acid determination. Bottles were stored at −20 °C until analyses.g for 15 min to remove sediment filter fragments. One milliliter of the supernatant was spectrophotometrically measured at 665 and 750 nm. To determinat the Chl-a content, we used the equation described by Marker et al., 1980, with a constant value of 12.99 [® , and salinity by a refractometer (Argent) in the Laboratory of Biogeochemistry of Greenhouse Gases (LABGEI) of the Pontificia Universidad Católica de Valparaíso, Valparaiso, Chile. Total Chl-a was quantified by methanol extraction and spectrophotometric determination. Each filter was incubated in 1.4 mL of hot methanol (65 °C) and bead-beaten with 1 mm glass beads (2 pulses of 1 min at 4000 rpm). The mix was incubated in the dark for 5 min at 65 °C and 15 min on ice. After the incubation, tubes were centrifuged at 15,000× of 12.99 . POC and® , and pH was measured using a pH indicator, Hydrion (9800), and a Spectral 0–14 Plastic pH Strip .Variables such as temperature, light intensity, and pH were measured in situ at the sampling sites. Temperature and light were recorded every five minutes using a data logger, the HOBO Pendanthttp://www.taraoceans-dataportal.org) and performed a distance-based analysis. We used MASH v1.1.1 , which uses the MinHash dimensionality-reduction technique to cluster the metagenomic sequences and calculates the Jaccard Index to report an all-against-all distance matrix for all metagenomic samples [In order to compare our metagenomes with global ocean data, we downloaded all of Tara Ocean’s surface samples , and reported read abundance for each subsystem level 1, 2, 3, and function ,56,57. T--meta), then obtained the protein sequences from the viral contigs using Prokka v1.12 (--kingdom viruses --metagenome), which uses Prodigal v2.6.3 for the prediction of open reading frames (ORFs) [cd-hit-2d -g 1 -n 4 -d 0, 60% identity, and 80% coverage) [https://github.com/Vasco-Varas/Bioload (1 November 2022). Then, we used the Patagonia viral proteins that were not similar to any TOV proteins to form new clusters with two or more ORFs (Patagonia Protein Clusters (PTGN PCs)), using CD-HIT under the same settings as the TOV .We assembled the quality-controlled reads from each viral sample (Vir S1/S2/S3) using SPAdes v3.8.0 (s (ORFs) ,63,64. Woverage) . We clasWe analyzed and visualized all results obtained through the methodologies described above using the R software along with the following packages: ampvis2, ggfortify, ggplot2, ggpubr, ggrepel, gridExtra, phyloseq, RColorBrewer, reshape2, taxize, tidyverse, and VennDiagram packages ,75,76,77In order to compare the diversity of the microbial and viral communities of Patagonia surface waters against marine global samples, we conducted a distance-based analysis using MASH distances. We used the Tara Oceans metagenomes due to the sampling density and cover of global oceans and found that Comau samples were more distinct at the genomic level than those from global oceans . MicrobiPlanktomarina temperata, which reached up to 30–40% relative abundance  is resistance to antibiotics and toxic compounds A. WithinFinally, we wanted to functionally profile the viral communities in the Comau Fjord. Environmental viromes, however, are difficult to profile due to intrinsic features of viruses, i.e., high substitution rates, large population sizes, high turnover, as well as practical issues such as a lower representation in databases than human or industry-related viruses. Thus, we decided to use the membership of protein clusters (PCs)  to gIn this study, we compared the microbial and viral communities of coastal surface waters in the Comau Fjord with those of the global oceans. We detected markedly different composition and structure in these Patagonian microbial and viral communities. Coastal and estuarine waters differ from the open ocean in their oceanographic characteristics as well as in their physical parameters. In particular, estuarine waters such as those of the Comau Fjord are subject to salinity changes due to glaciers melting and snow and precipitation (>5000 mm in the study site) , which mWe found similar patterns when looking at viral communities in the Comau Fjord. While viral communities were genetically distinct from those from the global oceans, a closer characterization showed that Comau viruses were cosmopolitan and had relatives mainly from the South Atlantic Ocean and Subarctic Pacific Ocean. Interestingly, higher-abundance viruses were taxonomically related to riverine and estuarine viruses from other continents, suggesting that environmental factors shape their evolution in a convergent fashion. For instance, Patagonia viruses were related to viruses described in the Chesapeake Bay, Delaware Coast, Guaymas Basin, Delaware River and Bay, Monterey Bay, and Columbia River. Similar to our findings, Sun et al. (2021) found a large number of diverse and abundant viruses without culturable representatives that were distinct among the Chesapeake Bay and Delaware viral communities and, in turn, highly differentiated from ocean viromes . SimilarEscherichia coli, highlighting the potential risk to human health [In this study, we also addressed the resistome of the microbial communities in the Comau Fjord, especially in relation to salmon farming. Functional profiles showed that Comau microbial communities were enriched in amino acid and protein metabolism genes, as well as other homeostatic functions such as carbohydrate and DNA metabolism, cofactors, vitamins, and others, as well as RNA metabolism (SEED Subsystem Level 1). Interestingly, a high proportion of virulence genes were assigned to resistance to antibiotics (SEED Subsystem Level 2), of which almost 40% belong to multidrug resistance efflux pumps, resistance to fluoroquinolones, and to beta-lactamases. Several studies have shown that salmon farming and aquaculture in general might promote the emergence and spread of antimicrobial resistance in sites far removed. For instance, Sha et al. (2014) analyzed 200 bacterial isolates from aquaculture and non-aquaculture sites in southern Chile and showed high levels of ARGs in both types of sites, of which some were able to transfer their resistance to n health . Similarn health  .We also specifically searched for ARGs in our dataset and compared it to regional samples from the South Atlantic, South Pacific, and Southern Oceans  to get insights as to the increased abundance or prevalence of ARGs in the Comau Fjord. Although our results were congruent with the literature, i.e., macrolide−lincosamide−streptogramin, multidrug, betalactams, and others ,89,90,91Viruses are important yet often neglected members of the ocean microbiome, which in part is derived from the difficulty in classifying their genes and genomes . Here, wIn this study, we tested whether Comau microbial and viral communities would be distinct in composition and function in comparison to those collected in global surveys; however, we expected they would bear the hallmarks of microbes from coastal and temperate regions. Our results support this hypothesis, as the Comau Fjord microbial and viral communities are diverse and different from those sampled in global ocean surveys. Similarly, most published global ocean surveys are densely sampled in regions other than cold-water and high-latitude oceans, which would partially explain the novelty of the Comau Fjord and locations alike. We also hypothesized that Comau microbial communities will be functionally enriched in ARGs in general and in those related to salmon farming. This was partially confirmed by our data since we were able to find genetic determinants of resistance against florfenicol and oxytetracycline . However, our study presents limitations. More dense sampling with a spatial and temporal design might be able to better capture any signal of disturbance from salmon farming. In addition, microbes attached to particles as well as sediment samples might yield a higher resolution picture of the anthropogenic disturbance caused by salmon farming. Finally, a meta-analysis of estuarine waters, fjords, and channels with comparable methodologies might provide further insight as to the effect of anthropogenic disturbances on the microbial and viral communities of the water column.Since most human activities occur in coastal areas, such as bays, sounds, fjords, and estuaries, they represent a unique ecosystem that needs to be further studied to understand the interplay between human and environmental health. In particular, determining whether microbial communities are resistant or resilient to disturbances of anthropogenic origin, i.e., diesel contamination and antibiotics, will allow us to understand to what extent their functions will also be affected in the future.