The objective is to find whether serum uric acid (SUA) levels are associated with the progression of chronic kidney disease (CKD) remains uncertain, and follow-up data among the elderly population are relatively lacking, especially in China. The aim of the present study was to reveal the association between baseline SUA levels, changes in SUA levels, and renal failure in Chinese elderly adults. 2. We used multiple linear and logistic regressions to compare the risk of renal dysfunction among the different SUA level groups.  In this retrospective cohort study, 425 subjects (age range 71–100 years) were analyzed and divided into quartiles based on baseline SUA levels . CKD was defined as estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73 mvs. 1.79 , 3.4 , and 6.79 , respectively; P for linear trend <0.001), and a per unit increase in baseline SUA levels gave an OR of 1.76  for renal failure. At the same time, a change in SUA levels had a stronger inverse correlation with a change in eGFR in females  than in males . In a linear regression analysis, a 1 mg/dl increase in SUA levels was associated with an additional 1.25  ml/min/1.73 m2 decrease in eGFR over a 3-year period.  The prevalence of hyperuricemia was 24.24% in the elderly subjects. In the multivariable analysis, the odds ratio (OR) for the development of CKD increased with the increase in SUA quartiles at baseline (1.00  Elevated baseline SUA levels and changes in SUA levels were associated with a decline in eGFR and an increased risk of CKD in an elderly Chinese population. Chronic kidney disease (CKD) has become a global public health problem. In China, patients with CKD account for 4.86% of all hospitalized patients, and their median cost and length of hospital stay are higher than those for patients without CKD . Among tSerum uric acid (SUA) is generated from the metabolism of purine nucleotides, and approximately two-thirds of SUA is excreted via the kidneys . Over thAlmost all of these studies were focused on adults, and follow-up data among the elderly population are relatively lacking, especially in China. In a cohort study including 1410 elderly residents from Beijing, Zhang reported an adjusted OR of 1.19 in the association between incident CKD and each 1 mg/dl increase in the SUA level . Another2; gout; malignancies; acute cardiovascular and cerebrovascular disease; infectious disease; or other life-threatening diseases. Another 29 subjects were excluded due to loss to follow-up. Consequently, a total of 425 subjects  were enrolled in this study. The Zhejiang Provincial People's Hospital Ethics Committee approved the protocol for this study (2020QT179).All participants in this study received two comprehensive health examinations in both 2018 and 2021, which were conducted at the Zhejiang Province People's Hospital. We excluded 38 subjects who had used diuretics or urate-lowering drugs at baseline and 21 subjects for having a past history of the following: a baseline eGFR <15 ml/min/1.73 m2). Blood pressures were measured by using a standard sphygmomanometer after the subjects rested for 15 minutes.Every subject was required to complete a questionnaire including information on demographic characteristics and medical history. After an overnight fast, they were examined in the morning by trained nurses. The concentrations of SUA, serum albumin, fasting plasma glucose, hemoglobin A1c (HbA1c), hemoglobin, serum creatinine (Scr), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and C-reactive protein (CRP) were measured by using a 717A automatic biochemical analyzer (Hitachi H7600). Urine creatinine (Ucr) and urine albumin (UALB) were measured through an automatic urine analyzer (Sysmex AX4280) and an automatic protein analyzer (Siemens BNII). The urinary albumin-to-creatinine ratio  was calculated as UALB/Ucr. Body mass index (BMI) was defined as body weight  divided by the square of height  was calculated by the CKD-Epidemiology Collaboration (CKD-EPI) equation [2) [2018-eGFR2021 and ∆SUA= SUA2018-SUA2021. Hypertension was defined as a systolic or diastolic blood pressure ≥140 or ≥90 mmHg, respectively, or the current use of antihypertensive drugs [HUA was defined as SUA>7 mg/dl in men and >6 mg/dl in women with a normal purine diet. All subjects were divided into quartiles based on their baseline SUA levels . The estimated glomerular filtration rate (eGFR) (ml/min/1.73 mequation . CKD wastion [2) , and albtion [2) . The chave drugs .T test and chi-square test, respectively. Statistical differences among the groups were compared with a one-way analysis of variance (ANOVA) or the Kruskal–Wallis test. In further analyses, multivariate logistic regression analysis was used to calculate odds ratios (ORs) between the baseline SUA level, changes in SUA levels, and CKD, after adjustment for confounding variables. Values with a P < 0.05 were used to define statistical significance.All analyses were performed by SPSS v. 22. Continuous variables are reported as the means±standard deviations or interquartile ranges (IQRs), and categorical variables are reported as percentages. Continuous and categorical variables were compared by the P < 0.001). Among all 425 subjects, the prevalence of diabetes, hypertension, and chronic cardiovascular disease was 36.24%, 80.47%, and 60.24%, respectively.The prevalence of HUA was 24.24% in the total population, and the prevalence of HUA was higher in females than in males , while the mean values of baseline eGFR, follow-up eGFR, TC, and HDL-C decreased significantly (P < 0.05). Pearson's correlation coefficient showed that the baseline eGFR was inversely correlated with the SUA level at baseline .Subjects were divided into quartiles (Q1-Q4) by SUA levels at baseline, and the baseline characteristics are presented in P for linear trend<0.001) .In Model 1, the OR for the progression of CKD increased as the quartiles for baseline SUA levels increased from the first to fourth quartiles  . In Moder = −0.187, P < 0.01) and females . After adjusting for confounding factors , we found that a 1 mg/dl increase in the SUA level over 3 years was connected with an additional 1.25 ml/min/1.73 m2 decline in eGFR  for individuals in the fourth SUA quartile (>5.1 mg/dl) compared to those in the first quartile [In our study, participants in the fourth SUA quartile were 6.79 times more likely to develop CKD than those in the first quartile, and a per unit increase in the baseline SUA level indicated an OR of 1.76  for renal failure. In addition, the OR for CKD significantly increased with baseline SUA levels, even within the normal range . This indicated that a mild elevation within the normal range of SUA might be a risk factor for renal dysfunction. Our findings were in accordance with those of previous studies –14,20. Iquartile –14. The 2 decrease in eGFR during the 3-year follow-up after adjusting for possible factors. Consistently, Ye M et al. revealed that the time-mean SUA could increase the risk of developing CKD by 6.32-fold [In the analysis of the longitudinal data, we observed that changes in SUA levels were inversely correlated with changes in eGFR, and a 1 mg/dl increase in the SUA level was associated with an additional 1.25 ml/min/1.73 m.32-fold . A retro.32-fold . In addi.32-fold . Additio.32-fold . Thus, wE et al. revealed that age-dependent UA increases were markedly different in males and females, with the latter showing a steeper trend [Furthermore, we found that sex differences might play an important role in the association. Musacchio er trend . In a prer trend . In our er trend , 26.However, the link between uric acid and kidney failure remains controversial. In a study by Chang YH et al., uric acid played a pathogenic role only when the value exceeded 6.3 mg/dl, and a lower uric acid level might be beneficial to the improvement of renal function in participants with diabetic nephropathy . Based oA series of experimental studies proposed several possible underlying mechanisms –32: (1) As a retrospective cohort study, the present study had several limitations. First, the small sample size and the retrospective nature of the study may weaken the potential causality between SUA levels and CKD. Second, we could not eliminate the possible effects of medication that affected uric acid metabolism on the present findings. Third, we had no information about the family history of kidney disease or underlying kidney disease, which should be obtained by renal biopsy and ultrasonography. Finally, the conclusions of this study are not applicable in other ethnic populations because of the sample's demographics.This study demonstrated that elevated baseline SUA levels and changes in SUA levels were associated with increased odds of CKD and declining eGFR among elderly Chinese individuals after adjusting for possible confounding factors. Early and appropriate management of SUA levels might slow the development of future declines in eGFR, but we need further large-scale prospective trials to elucidate this issue.


This project aimed to evaluate the effects of both different additives and filling methods on nutritive quality, fermentation profile, and in situ digestibility of whole plant corn silage.3. All silos were weighed and stored in lab at ambient temperature.Whole plant corn forage harvested at 26.72% dry matter (DM) was chopped and treated with two filling methods, i) fill silos at one time (F1), ii) fill silos at three times (F3), packing samples into one/three silo capacity at the first day, another one/three capacity at the second day, then one/three at the third day, three replicates. For each replicate, samples were treated with three additives, i) control , ii) Sila-Max , and iii) Sila-Mix . With three replicates of each secondary treatment, there were nine silos, 54 silos in total. Each silo had a packing density of 137.61 kg of DM/mAfter 60 d of ensiling, all items showed good silage fermentation under MAX filled one time or three times (p<0.01). Higher silage quality for all additives was obtained at filling one time than that filled three times (p<0.01). The highest DM and lowest DM loss rate (DMLR) occurred to MAX treatment at two filling methods (p<0.01); Digestibility of acid detergent fiber, neutral detergent fiber (NDF), and curde protein had the same results as silage quality (p<0.01). Yield of digestible DM and digestible NDF also showed higher value under MAX especially for filling one time (p<0.05).All corn silages showed good fermentation attributes (pH<4.0). The forage filled one time had higher silage quality than that filled three times (p<0.01). MAX with homofermentative lactic acid bacteria enhanced the lactic acid fermentation, silage quality and nutrient digestibility, and so improved the digestible nutrient yield. Acetobacter pasteurianus [China is an agricultural country with abundant straw resources. More than 8.4 billion tons of straw is produced every year . In tradeurianus . For whiL. plantarum, dried E. faecium, P. acidilacitici, dried P. acidipropionici, fruct-oligosaccharide and starch, with adding dosage of 2.5 g/t fresh forage, dissolved in 4 L deionized water, then sprayed on forage evenly, supplied a final application rate of 2.5×108 of lactic acid bacteria (LAB)/kg fresh forage; Mix contained the following active ingredients: lactic acid (LA), dried L. plantarum, dried P. acidilactici, dried E. faecium, dried P. acidipropionici, dried Bacillus subtilis, dried Aspergillus, fructo-oligosaccharide, starch, iron oxide and cobalt-lactic, with adding dosage of 1.0 kg/t fresh forage, the same method on forage, supplied a final application rate of 1.8×106 of LAB/kg fresh forage. The same amount of deionized water was also applied to CTRL. With three replicates of each secondary treatment, there were nine silos, 54 silos in total. Each silo had a packing density of 137.61 kg of DM/m3. All silos were weighed and stored in lab at ambient temperature. After 60 d of storage, the silos were weighed, opened, and samples were collected for measurement of nutrients concentrations, nutrients digestibility, pH, ammonia, volatile fatty acid (VFA), LA, and lowest DM loss rate (DMLR).Whole plant corn forage  at dough stage maturity, harvested by a forage chopper  on October 1, 2015, on a dairy farm in Lintao county, Gansu, China was chopped to a theoretical length of cut of 1 cm . Samples3-N was measured using phenol-hypochlorite method according to Broderick and Kang [18 column and G132B ultraviolet fluorescence detector . The total VFA concentrations include LA concentrations.Both fresh juice and silage juice were extracted by blending 10 g forage (wet basis) in 90 mL of distilled water through double-layered cheesecloth and filter paper . The mixture was stored for 24 h at 4°C in a refrigerator , and theand Kang . LA and 2O%–CP%–CF%–Ash%–EE%. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to the methods of AOAC [Both fresh and silage samples were dried by placing them in a forced-air oven at 65°C for 48 h to determine DM and then were ground to pass through a 1 mm screen using a grinding mill  and stored for analysis . The cru of AOAC . Hemicel3-N (% TN).All the chemical analyses were conducted in triplicate, and the results were expressed on a DM basis except for DM content (% fresh matter) and NHad libitum access to drinking water.Six Dorset×Small Tail Sheep  rams with ruminal cannula and an average of 59.2±6.3 kg (mean±standard deviation) body weight were used to measure nutrients ruminal digestion. All animals were cared for according to the Chinese standards for the use and care of research animals . The total mixed ration (TMR) was formulated to meet the nutrient requirements of a ram gaining 50 g/d according to the mutton sheep breeding standards (NY / T816-2004), Agricultural Industry Standards of the People’s Republic of China. The forage to concentrate ratio was approximately 70:30 (1.6 kg/d corn straw silage and concentrate supplement). The ingredient and nutrient compositions of the TMR are given in The DM, NDF, HC, and ADF disappearance were mea sured according to the standardized procedures of Harazim and Paveleck . Five grYield of DM (g/kg) was calculated according to DM content, yield of NDF (g/kg) was determined by NDF concentration. Yield of digestible DM  and digestible NDF  were calculated according to DM and NDF yields and their digestibility, respectively.All data were subjected to least-squares analysis of variance using the PROC general linear model procedure of SAS  . The modSignificant differences were found on corn silage fermentation characteristics between different treatments at two filling times. All items showed good silage fermentation results under MAX treatment at either filling one time or three times (p<0.01). With respect to filling time, there was higher silage quality under all additives treated with filling one time than those treated with three times (p<0.01). There were significant differences for all fermentation indexes under interactions of FM×ADD (p<0.01) except propionic acid. Although the pH was similar among treatments under different filling methods, it was lower under MAX and MIX than that under CTRL for F1 , but there was the same value of 3.23 under different ADD treated with F3. The content of LA treated with MAX improved by 24.09% and 0.62% than CTRL and MIX at F1 while improved by 24.19% and 4.39% at F3 respectively. For acetic acid, propionic acid and butyrate acid, all of them treated with MAX and MIX were lower than those treated with CTRL either at F1 or F3. And the value of acetic acid/propionic acid treated with MAX was significantly higher than that of MIX and CTRL at two FMs . Except The ADF digestibility (ADFD), NDF digestibility (NDFD), and CP digestibility (CPD) of silage treated with MAX at two filling times were higher than those treated with CTRL and MIX, but they were not different between treatments except for CPD. For each treatment, filling times affected the results significantly (p<0.01); ADFD, NDFD, and CPD of each additive filled one time were all higher than those filled three times (p<0.01). The interaction between FM and ADD showed no differences . The yieAfter 60 d of ensiling, all corn silage showed excellent fermentation characteristics, which was indicated by dominant LA content as well as negligible propionic and butyric acid. This change can be ascribed to the presence of a sufficient fermentable substrate of the whole corn plant in the milk stage to the ripeness stage as the test material used in the experiment. But further research is still required to clarify why there was higher LA percent compared to CTRL under MAX at F3 than that of F1. Water-soluble carbohydrate (WSC) content along with the activity of epiphytic LAB determines the rate of decline in pH during the early stages of ensiling, which is important for producing stable silage . WoolforThe literature review published by Muck and Kung  establisUsually, silage fermentation produces volatile compounds, which will often cause the apparent DM% of silage to decrease compared to fresh material . In presin vitro DMD and NDFD of corn silages and found that DMD improved with some inoculants, whereas NDFD did not. The authors concluded that this effect might be due to some solubilization of HC during ensiling, which improved DMD but did not change or even decreased the digestibility of the residual NDF. The present study had lower CPD after treatment with MAX, which likely contributed to the acid produced by LAB fermentation reducing the apparent digestibility of CP in the rumen and increasing the proportion of bypass protein flowing to the duodenum. This result is consistent with the data of Broderick [In our study, although no significant differences in DMD, ADFD, and NDFD with the exception of CPD among treatments were observed, MAX slightly increased DMD, ADFD, and NDFD  and therroderick , who repKnowledge about the effects of ensiling conditions, e.g., delayed sealing and air infiltration during the ensiling process, is still rare . In farmAdditionally, compared to silage filling times, silage filled one time had higher DM and lower DMLR, EE and Ash (p< 0.01). Although other nutrient indexes also have consistent results, they showed no biological significance . TherefoAfter 60 d of ensiling, due to sufficient fermentable substrate and epiphytic LAB in whole plant corn, all corn silages showed good fermentation attributes (pH<4.0). MAX with homofermentative LAB enhanced the LA fermentation, silage quality and nutrient digestibility, compared to CTRL and MIX, and the digestible nutrient yield was also improved, which showed that MAX can be popularized and used in farm corn silage practice in northern China. Silages treated with all additives filled one time, compared to samples filled three times, attained good fermentation qualities; this finding indicates that residual oxygen during ensiling can impair the growth rate of some homofermentative bacteria and lead to a decline in LA concentration and silage quality. Therefore, filling the silo one time is recommended in ensiling process if the conditions are suitable.


There are no effective therapeutic options for locally advanced head and neck squamous cell carcinoma (HNSCC). Additionally, there is no standard therapy for patients subjected to multiple lines of treatment. Angiogenesis plays a key role in tumor growth and metastasis. Therefore, inhibition of tumor angiogenesis is an important strategy for tumor therapy. Apatinib is a novel tyrosine kinase inhibitor that inhibits angiogenesis by targeting vascular endothelial growth factor receptor-2 (VEGFR-2). The effect of apatinib on HNSCC has not been clearly established. In this study, we administered apatinib in combination with anti-epidermal growth factor receptor (EGFR) targeted and systemic chemotherapy for the treatment of oral cancer and to achieve better disease outcomes. To avoid fatal bleeding, after achieving good clinical outcomes, the follow-up treatment plan was adjusted. The efficacy of apatinib combined with anti-EGFR targeted and systemic chemotherapy for the treatment of oral cancer has not been previously reported. Our findings show the therapeutic potential of apatinib for advanced HNSCC patients with multiple lines of chemotherapy, especially for patients with large neck masses. It is the most common SCC of the head and neck. Annually, there are about 350,000 new cases and over 170,000 mortalities associated with oral cancer, globally accounting for 2.0 and 1.9% of new morbidity and mortality cases, respectively [23 × 6 cm3 × 7.5 cm3 on the right neck that invaded adjacent muscles and skin, with an irregular shape, unclear boundaries, and poor movement. There was an ulcer of about 1.5 cm2 × 2 cm2 in the buccal mucosa. Her routine blood and blood biochemical parameters were normal. Her serum SCC antigen level was 2.4 ng/mL (reference range: <1.5 ng/mL), while carcinoembryonic antigen (CEA) level was within normal ranges. The pathological biopsy , which was performed in the dental outpatient clinic, revealed highly differentiated SCC. Immunohistochemistry staining indicated VEGF (+). B-ultrasound revealed a solid cystic mass in the right submandibular with multiple lymphadenopathy. The positron emission tomography computed tomography (PET-CT) scan revealed a deep cystic solid mass in the posterior margin of the right parotid gland with uneven fluorodeoxyglucose uptake. The mass involving surrounding tissues, multiple metastatic cervical lymph nodes, with no clear boundary with the right common carotid artery and no metastasis in other parts. The diagnosis was oral SCC at clinical stage T2N3bM0 (IVB). The patient’s body surface area (BSA) was 1.53 m2. The first-line chemotherapeutic option was intravenous paclitaxel (135 mg/m2) on day 1 and cisplatin (30 mg/m2) on day 1–3. However, there was no decrease in neck mass volume after one cycle. Magnetic resonance imaging (MRI) revealed a soft tissue mass with a volume of about 7.5 cm3 × 5.5 cm3 × 7 cm3 on the right neck that invaded adjacent muscles and skin and multiple enlarged cervical lymph nodes (A 56-year-old woman presented with a 3-month history of a painful oral ulcer and local mass in the right cheek. The patient denied any history of hypertension, diabetes, coronary heart disease, smoking, drinking, or hereditary disorders. Physical examination revealed a mass of about 8 cmph nodes ; Table 1ph nodes ; Table 1ph nodes . After oph nodes . After aph nodes ; Table 1Ethics and consent statements: This study was approved by the ethics committee of Anhui Provincial Cancer Hospital, and written informed consent was obtained from the patient.3Due to the atypical symptoms and signs of oral cancer, more than 60% of patients are diagnosed at stage III or IV. They lose the chance for surgery and can only be comprehensively treated by radiotherapy and chemotherapy . NeoadjuTumor angiogenesis plays a key role in tumor growth and metastasis. For continuous cell proliferation, tumor tissues rely on oxygen and nutrients provided by neovascularization. The VEGF/VEGFR signaling pathway is considered the most critical step in tumor angiogenesis. VEGFR inhibitors can bind VEGF, which suppresses the binding of VEGF to VEGFR on the surface of vascular endothelial cells, thereby inhibiting biological effects, including tumor micro angiogenesis, invasion, and metastasis . ApatiniApatinib has been clinically used for various late-stage cancers, including hepatocellular carcinoma , non-smaCommon side effects of apatinib, including hypertension, oral ulcer, hand-foot syndrome, proteinuria, and fatigue, are generally manageable and acceptable. The patient did not present these side effects, which were attributed to the short-term application of apatinib. The possible reasons for pulmonary infection in this patient were: i. Poor immunity due to the effects of cancer, making her susceptible to lung infections; ii. Pulmonary metastases, pleural effusions, and other coinfections in the lungs; and iii. Bone marrow suppression and immune deficiency after chemotherapy, which led to pulmonary infections. A direct association between pulmonary infection and apatinib has not been reported. This patient was administered with oral apatinib for one month and did not present any symptoms of pulmonary infection during this period; therefore, it was very unlikely that apatinib caused pulmonary infection. Studies should determine whether apatinib can cause pulmonary infections.In this case, apatinib played a rapid and overwhelming role in tumor control. However, for clinical applications of apatinib, various considerations should be paid attention to. First, apatinib is effective for advanced HNSCC patients with multiple lines of chemotherapy treatment, especially in patients with large neck masses. Apatinib can be used in combination with anti-EGFR targeted and systemic chemotherapy, as described in our case. However, the role of apatinib in HNSCC should be confirmed in large-scale prospective clinical trials. Second, there is a need to establish the appropriate cohort of patients to receive apatinib. HNSCC patients with tumor invasions of large blood vessels have a high risk of fatal bleeding; therefore, apatinib should be used with caution. Third, tumor regression and necrosis are closely observed during the treatment of apatinib. Meanwhile, the coagulation function should be regularly monitored, and the treatment plans should be adjusted in time to avoid the risk of fatal bleeding.In conclusion, apatinib combined with anti-EGFR targeted therapies and chemotherapy may be effective for advanced HNSCC patients, especially for patients with large neck masses to avoid the occurrence of fatal bleeding. However, the efficacy and safety for apatinib should be confirmed by multicenter large-scale prospective clinical trials, which may provide an effective treatment modality for HNSCC patients.HNSCChead and neck squamous cell carcinomaVEGFR-2vascular endothelial growth factor receptor-2EGFRepidermal growth factor receptorSCCsquamous cell carcinomaCEAcarcinoembryonic antigenPET-CTpositron emission tomography computed tomographyBSAbody surface areaMRImagnetic resonance imagingRECISTresponse evaluation criteria in solid tumorsSDstable diseasePFSprogression-free survivalOSoverall survivalCFDAChina Food and Drug Administration


Gambusia affinis across parallel thermal gradients in New Zealand (NZ) and California (CA) to understand the influence of temperature on diets, morphology and stoichiometric phenotypes. Our results show that with increasing temperature in NZ, mosquitofish consumed more plant material, whereas in CA mosquitofish shifted towards increased consumption of invertebrate prey. In both regions, populations with plant-based diets had fuller guts, longer relative gut lengths, superior-orientated mouths and reduced body elemental %C and N/P. Together, our results show multiple pathways by which consumers may alter their feeding patterns with rising temperatures, and they suggest that warming-induced changes to resource availability may be the principal determinant of which pathway is taken.Rising temperatures may alter consumer diets through increased metabolic demand and altered resource availability. However, current theories assessing dietary shifts with warming do not account for a change in resource availability. It is unknown whether consumers will increase consumption rates or consume different resources to meet increased energy requirements and whether the dietary change will lead to associated variation in morphology and nutrient utilization. Here, we used populations of  In warmmaterial . The drimaterial ,6. Altermaterial –9.Current theories on dietary shifts with rising temperature rest on shifting consumer demand from a physiological response to temperature, but they do not consider other changes in food webs that also occur with warming. Importantly, the temperature rise will change resource availability as thermal limits for some species are exceeded, and colonization by new species occurs –12. As sShifts in resource availability and diet along thermal gradients should select for morphological traits which maximize resource use efficiency –20. For p skeletal stores [Body morphology and resource availability changes associated with increased temperatures may alter consumer elemental composition –28. Wherl stores . In addil stores ,29,31. TGambusia affinis) established from repeated recent invasions of springs that span parallel temperature gradients in New Zealand (NZ) and California (CA). We use a space for time substitution approach to explore how shifts in diet across the temperature gradients are associated with divergence in phenotypic traits  and body elemental composition. We hypothesized that with warmer temperatures, populations of Gambusia affinis would consume more plant-based materials as invertebrate resources decline and demand for carbon increases leading to longer guts, altered feeding traits , and a body elemental composition reflective of environmental resources and morphology Gambusia affinis (hereafter ‘Gambusia’) were introduced from populations in Texas, USA to CA, USA in the 1920s and to the North Island of NZ in the 1930s [Gambusia are live-bearers, reach high densities in the wild, and are found across a wide range of environmental conditions: salinity, temperature, pH and dissolved oxygen (DO) [Gambusia have a broad thermal niche, tolerating temperatures from freezing to approximately 40°C and reproduce in temperatures above approximately 16°C [he 1930s –35. Gambgen (DO) . Gambusiely 16°C . These oely 16°C –40.Gambusia from sites along parallel temperature gradients in NZ (19.2–37.7°C) and CA (18.8–38.9°C) , specific conductivity (mS cm−1) and pH . The springs were not chemically extreme, with conductivity values typical of non-geothermal waters in the regions, circumneutral pH and high levels of DO. The sites ranged from 1 to 263 km apart within each region and were not hydrologically connected on the surface . Temperature differences among these sites were not associated with spatial distance among sites . The presence of potential competitor and predator species was noted from visual surveys and netting carried out during site visits. Predator and competitor species were present in some sites , but most sites lacked heterospecific fishes, and their presence was not correlated with temperature  . We studears ago ,42.Tabl (b)Gambusia were captured using a 5 m seine (1.6 mm mesh) deployed at several locations. All Gambusia were immediately euthanized with MS-222 in CA or clove oil in NZ and transported back to the laboratory on ice and immediately frozen.In CA, we sampled between 30 May and 1 June 2016, and in NZ, we sampled from 8 February to 21 February 2017. At each site, 2 areas at each site, including different habitat types in proportion to their relative abundance. The contents of the D-net were pooled and preserved in 80% ethanol. Macroinvertebrates were identified to the lowest practical taxonomic unit  under 10–80× magnification and counted to give relative abundances of each taxon and taxa richness in each sample. Invertebrates were pooled and dried at 60°C for 48 h to measure overall invertebrate dry mass. Planorbid snails were excluded from dry mass measurements as these are too large to be consumed by Gambusia.Macroinvertebrate communities were surveyed at each of the sites via a standard protocol using a D-net (0.5 mm mesh) . We sampZooplankton were collected using a 63 µm mesh Wisconsin plankton net. At each site, this net was dragged horizontally under the water surface for 20 m, for a total volume of 13 l. All plankters were preserved in 80% ethanol. In the laboratory, zooplankters were enumerated and identified to the lowest possible taxonomic unit  under a 10–80× magnification microscope. (c)Gambusia from each of the 18 sites (n = 720). Gambusia were weighed (±1 mg), and lateral photographs were taken for body morphological measurements. We then removed the whole intestine of each Gambusia, cutting directly above the anus and immediately below the pharynx, uncoiled it and photographed it for length, and preserved it in 70% ethanol until gut content analysis. No other organs  were removed during dissection. Gambusia (less their guts) were dried at 60°C for 48 h before being ground for whole-body stoichiometric analysis (see below).We randomly selected 40  mature individual Gambusia using ImageJ [We measured the functional morphological traits relative gut length, eye size, eye height and mouth position on each g ImageJ ; these m (d)2 and counting how many 1 mm2 cells on the graticule were filled [Using the entire gut, individual gut contents were removed and placed onto a Petri dish with a 1 × 1 mm graticule. Contents were identified under an 80–100× microscope to the lowest taxonomic resolution possible. The proportion that each prey item contributed to total gut volume was visually estimated . The vole filled . Animal  (e)n = 360). Elemental C and N were measured using a vario EL cube elemental analyser. For each individual, we used approximately 5 mg of dried and ground tissue . For total P analysis, approximately 2–3 mg of dried and ground Gambusia tissue was ashed in a furnace at 550°C for 4 h. Combusted samples were digested by adding 10 ml of distilled water and 2 ml of 2 N HCl into each tube; tubes were then placed into an oven at 105°C for 2 h [Body elemental composition was measured on 20 mature individuals  from each of our 18 sites ( for 2 h . Followi for 2 h . (f) (i)Gambusia populations in NZ and CA . Only one site (FC in CA) had a known predator species  to compare models using the aictab function in ‘AICcmodavg’ version 2.3-1 [We used a model comparison approach to evaluate which factors  best-explained variation in traits (e.g. gut length) among  species . To simpe matrix . We conson 2.3-1 . We rankon 2.3-1 . (ii)i) was summarized for each population using a relative importance index (RIi). This index accounts for both frequencies of occurrence of a diet item (%Fi) and percentage volume in the gut (%Vi) [i was calculated as %Fi × %Vi.Variation in diet items (ut (%Vi) .2.1RIi= i data for each site using the Jaccard distance metric with temperature fit as an environmental gradient onto the ordination.We summarized variation in diet among populations using ordination by non-metric multidimensional scaling (NMDS). NMDS was carried out on the RIGambusia guts that were independent of gut length, we calculated gut fullness (GF) asV is the volume of food in the Gambusia gut (mm2) and GL is gut length (mm).To determine if there were differences in the volume of prey in Independent generalized linear models (GLMs) were fitted to data from NZ and CA to analyse trends in GF, with site temperature and sex as independent factors.i) and morphological traits  were related to site temperature. We separated all morphological trait analyses by sex due to sexual dimorphism in Gambusia [We used simple linear models to determine if trends in diet  for comparison of the dominant trends.For plots of RI (iii)Gambusia mass on %C, %N, %P, C/N, C/P and N/P. Data were split by sex as there is some expectation that stoichiometry may vary with sex [We used separate GLMs by both region and sex to understand the effects of temperature and with sex . We usedGambusia condition changed with temperature, we calculated individual condition factor (CF) [W is wet mass (g) and SL (cm) is the standard length. Five outlying  CF values were excluded from analysis. We used GLMs with data were split by region to understand the effects of temperature and sex on CF.To understand if tor (CF)  as2.3CF=10 transformed before analysis to meet the models' normality assumptions. All statistical analyses were performed using R v. 4.1.0, and NMDS ordinations were produced using ‘Vegan’ version 2.5-6 [Where appropriate, data were logon 2.5-6 ,55. All on 2.5-6 .. 3 (a)r2 = 0.651, p = 0.005; figure 1a), but there was no relationship between temperature and macroinvertebrate dry mass in CA . Across all sites, macroinvertebrate dry mass was about 2 X higher in CA  compared to NZ  and total species richness across all sites was higher in CA (n = 13) compared to NZ (n = 9). In both regions, all but one invertebrate was benthic (Dytiscidae in CA and Mesovelia in NZ). Zooplankton density showed no relationship to temperature (p = 0.787) in NZ, but declined as site temperature increased  in CA. Zooplankton density was 2.4 times lower across NZ sites when compared to CA. In both NZ (p = 0.524) and CA (p = 0.777), there were no distinct patterns of change in macroinvertebrate community composition with temperature .Macroinvertebrate dry mass decreased 27-fold from our coolest to our warmest site in NZ ( (b)b,e; electronic supplementary material, figure S4). In NZ, RIi values shifted from invertebrate dominated to algal and detritus-dominated diet with temperature , while we observed no significant trend in diet with temperature CA (p = 0.126). Similarly, in NZ, NMDS ordination showed an increasing dominance of detritus, algae and terrestrial invertebrates in Gambusia diets with temperature . In CA, NMDS trends were less clear, and differences were not related to temperature (p = 0.576).In NZ and CA, there were shifts in diet at higher temperatures; however, these trends were opposite to one another and were stronger in NZ compared to CA (figure 1 (c)Modelling indicated that temperature was the best predictor of relative gut length and stoichiometric trends in CA and NZ . Temperature was the best predictor of mouth position and one of the best predictors of eye size in CA. By contrast, in NZ, body morphological traits were most frequently explained by competitor presence. Specific conductivity best explained GF and CF in CA, whereas in NZ spatial distance best explained GF and DO, and specific conductivity best explained CF.p < 0.0001) and CA (p = 0.002), and within each region, both sexes showed similar patterns of gut length change with temperature. However, the direction of gut length change with temperature differed between regions . In NZ, where diet composition shifted to plant-based diets, relative gut length increased with temperature , with the degree of change being slightly stronger for females than males .Females had longer relative gut lengths compared to males in NZ . There was a similar tendency for relative gut length to increase with algae and detritus in Gambusia diets in CA, although this trend was not statistically significant .While temperature and gut length trends were divergent, both regions showed similar relationships between gut length and plant material in t = −5.73, p < 0.0001) and females . Whereas in NZ, mouth position was higher with temperature, but this trend was significant for males  but not females . In both regions, eye size increased with temperature. In CA, the increase in eye size with temperature was significant for males  and females . In NZ, this trend was significant for males  but not females . There were no significant trends in eye position in NZ or CA for either sex .Body morphology measurements showed a divergence in mouth position with temperature between NZ and CA, whereas the presence of competitor species at our study sites did not significantly explain these measurements . In CA, mouth position was lower at higher temperatures in males . Temperature was the key determinant of %C, %P, C/P and N/P in NZ, and %N and C/N in CA. In NZ and CA, temperature affected Gambusia elemental composition, where populations with plant-based diets had reduced body elemental %C and N/P. In NZ, %C  decreased with increasing site temperature  and %N , which decreased with increasing temperature in NZ. In NZ, male N/P decreased with increasing temperature , and there was a significant interaction between temperature and mass . For females, there was no relationship between N/P and temperature  or mass . There was no significant change in %P across the temperature gradient and dry mass was unrelated to elemental nutrient percentages (p > 0.05) in NZ. Whereas, in CA %C  and N/P increased  with increasing site temperature, but these patterns were only evident for males. In CA, %N increased with mass for females but not for males . For females in CA, there was an interaction between temperature and mass with %N , and there was a weak interaction between temperature and mass with N/P .perature . We foun. 4Increased temperature is predicted to increase consumer energy demand and shift the composition of available resources, thereby altering consumer diets and potentially consumer trophic traits ,20,57,58 (a)Gambusia resource use differences in this study. In NZ, macroinvertebrate food resources were scarcer than in CA at cool temperatures and further decreased with increased temperature, probably necessitating a switch to a diet dominated by algal and detrital matter. Further, warm populations in NZ had a greater volume of food in their guts in comparison to cool populations, suggesting compensatory feeding with temperature rise to meet the higher metabolic energy demands associated with warmer temperatures [Gambusia may alter their feeding patterns with rising temperatures, as Gambusia either forage for invertebrate prey or consume greater volumes of plant material to meet energy demands.Change in prey community composition and biomass is likely to occur with temperature change, as thermal limits for species are exceeded, and colonization by new species occurs –12. Sucheratures . Trends eratures ,59. The eratures , and thieratures , our dateratures . Overall (b)Gambusia diet in NZ, this relationship was not significant in CA. Invertebrate resources were not as limited in CA, so Gambusia may be more reliant on invertebrate materials at all temperatures when compared to Gambusia in NZ, where animal resources were scarce at warm sites. The change in gut length with food resources suggests that Gambusia can adjust their phenotype to meet their energetic demands. Plasticity in gut morphology is common and may occur with diet changes or with fasting [et al. [Poecilia reticulata) did not alter the development of gut length, and gut length remained longest in populations with a plant-based diet, suggesting local adaptation [Gambusia here is unknown; however, the presence of parallel patterns of gut length with dietary shifts in NZ and CA highlights the functional significance of morphological changes in Gambusia.Like other studies, our data show an increase in gut length with a shift toward plant-based diets ,25,62–65 fasting ,62,65–67 [et al.  found evaptation . The mecGambusia where animal prey are abundant. When benthic animal prey were less available, mouth position was more superior. Long filamentous algae were common in warmer sites in NZ , so fish may be feeding in the water column rather than on algae attached to the benthos. Alternatively, a superior mouth position may enhance surface feeding on terrestrial prey, which is typical of Poeciliidae [The morphological traits, mouth position and eye size also responded to temperature. Mouth position responded in the same way to dietary shifts in NZ and CA, where mouth position became more superior (e.g. higher on the head) as diets became more plant-based, and mouth position became more sub-terminal (e.g. lower on the head) where diets were predominately animal-based. Many of the animal resources identified in our field sites were benthic; thus, our data suggest feeding on the benthos is a common feeding strategy for ciliidae .In both regions, an increase in temperature was associated with larger eye size. In CA, increasing eye size with rising temperature may enhance visual acuity for finding prey ,69. Howe (c)Gambusia elemental composition was influenced by both site temperature and sex. We found no other studies which have reported Gambusia elemental composition values; however, those found here were within the range of values found in other species in response to different biotic and abiotic factors [Gambusia have a longer lifespan than males, and Gambusia are sexually dimorphic  [ factors ,30,70,71aturity) –74. Consaturity) . RegardlGambusia elemental composition. While diet shifted in the opposite direction with temperature in NZ and CA, elemental composition tracked diet shift in the same manner. In NZ, %C, C/N and N/P decreased as temperature increased, and in CA, %C and N/P increased with temperature. Reduced %C and N/P with a plant-based diet in both regions indicates a reduction in lipid stores and muscle tissue, and thus an overall decrease in fish condition [Dietary shifts towards plant and detrital material were consistently associated with increased N and reduced C in ondition .Gambusia [Despite the different patterns in stoichiometric proxies for condition between regions, our other condition metric—the CF—showed decreases with temperature in both regions. Although this pattern is typical of many fish species, including Gambusia , it shou. 5Gambusia ate more algae. As the temperature increased in CA, invertebrate prey remained stable and Gambusia ate more animals. A plant-based diet was associated with comparatively longer and fuller guts, a more superior mouth position, and decreased elemental C and N/P in body tissue in both regions. Thus, to understand how consumer diet, elemental composition and morphological traits change with increased temperature, we need to understand how temperature influences resource availability and that may not play out the same in different places.Using a space for time substitution approach, we observed that increased temperature was associated with altered resource availability. This was reflected in the diet of fish, with predictable changes in morphology and body elemental composition. Metabolic rates of mosquitofish are higher in the warmer systems , and theClick here for additional data file.


In the withheld validation dataset, these genes could delineate patients with IAs from controls, as the majority of them still had the same direction of expression difference. Bioinformatics analyses by gene ontology enrichment analysis and Ingenuity Pathway Analysis (IPA) demonstrated enrichment of structural regulation processes, intracellular signaling function, regulation of ion transport, and cell adhesion. IPA analysis showed that these processes were likely coordinated through NF-kB, cytokine signaling, growth factors, and TNF activity. Correlation analysis with aneurysm size and risk assessment metrics showed that 4/54 genes were associated with rupture risk. These findings highlight the potential to develop predictive biomarkers from PBMCs to identify patients harboring IAs.Peripheral blood mononuclear cells (PBMCs) play an important role in the inflammation that accompanies intracranial aneurysm (IA) pathophysiology. We hypothesized that PBMCs have different transcriptional profiles in patients harboring IAs as compared to IA-free controls, which could be the basis for potential blood-based biomarkers for the disease. To test this, we isolated PBMC RNA from whole blood of 52 subjects  and performed next-generation RNA sequencing to obtain their transcriptomes. In a randomly assigned discovery cohort of  Intracranial aneurysms (IAs) are pathological outpouchings within cerebral vasculature that are present in about 3–6% of the general population ,2. The rClinical reports and animal model studies have shown that IA pathology is dominated by inflammation ,12,13,14In this study, we hypothesized that peripheral blood mononuclear cells (PBMCs), which are predominantly composed of monocytes, T cells, and B cells, have different transcriptional profiles in patients with IAs as compared to IA-free patients. Our rationale was that ongoing inflammatory responses at the IA tissue level could lead to peripheral cellular activation and chemoattraction of PBMCs, which has also been observed for circulating neutrophils in IA. Sabatino et al. demonstrated the initial feasibility of this concept in 2013 . Using aTo overcome these challenges and test our hypothesis, we performed transcriptome profiling on PBMCs in patients with and without IAs (confirmed on angiography). We used next-generation RNA sequencing to identify an IA-associated expression signature in PBMC transcriptomes. We further assessed if the IA-associated signature could distinguish patients with and without IA in a small, independent cohort of patients. Gene ontology analysis and physiological pathway modeling were used to determine the biological function of differentially expressed transcripts. Results from this study could motivate future efforts towards developing blood-based biomarkers and shed light on the pathophysiology of aneurysms.This study was approved by the University at Buffalo’s Human Research Institutional Review Board . Written informed consent was obtained from all subjects prior to sample collection and the study was carried out in accordance with the approved protocol. Patients at Gates Vascular Institute  receiving cerebral digital subtraction angiography (DSA) with and without IA diagnosis were enrolled in this study. Indications for DSA included confirmation of IAs detected on noninvasive imaging or follow-up imaging of previously detected IAs for the IA group, or to identify presence or absence of vascular disease  for the control group. Patients who consented to participate in this study were over 18 years old, English-speaking, and had not previously been treated for IA. Patients who were pregnant, had a fever (>100 °F), recently had invasive surgery, were receiving chemotherapy treatments, had autoimmune disease, or were on immunomodulating drugs were excluded. Information about patient’s history and comorbidities was collected from medical records.Between December 2013 and September 2018, we collected whole blood samples from patients receiving cerebral DSA  ,7,8,29. Whole blood was drawn into 10-mL citrated CPT tubes  from the femoral access sheath. Cell separation procedures occurred within 1 h from when the specimens were drawn. PBMCs were separated from granulocytes and red blood cells (RBCs) by density gradient centrifugation. The PBMCs were washed with RBC lysis buffer, pelleted and then disrupted with TRIzol reagent  and stored at −80 °C until further processing. RNA was extracted using the TRIzol kit, according to the manufacturer’s instructions. Trace amounts of DNA were removed by DNase I  treatment. RNA was purified using the RNeasy MinElute Cleanup Kit  and suspended in RNase-free water. The purity and concentration of RNA in each sample were measured by absorbance at 260 nm on a NanoDrop 2000 spectrophotometer , and 400 ng of RNA was sent to the University at Buffalo’s Next-Generation Sequencing and Expression Analysis Core facility for further quality control. Precise RNA concentration was measured at the core facility via the Quant-iT RiboGreen Assay  with a TBS-380 Fluorometer . The quality of the RNA samples was measured with an Agilent 2100 BioAnalyzer RNA 6000 Pico Chip . RNA samples of acceptable purity (260/280 ratio of ≥1.9) and integrity (RQN  ≥  8.0) were considered for RNA sequencing.RNA libraries were prepared using the Illumina TruSeq stranded total RNA gold kit . All samples underwent 100-cycle, dual-read sequencing in the Illumina NovaSeq6000 System  on a single flow cell and were demultiplexed with Bcl2Fastq. Per-cycle basecall files generated by the NovaSeq6000 were converted to pre-read FASTQ files using bclfastq version 2.20.0.422 using default parameters. The quality of the sequencing was reviewed using FastQC version 0.11.5. Potential contamination was detected using FastQ Screen version 0.11.1. No adapter sequences were detected, so no trimming was performed. Genomic alignments were performed using HISAT2 version 2.1.0 using default parameters. NCBI reference GRCh38 was used for the reference genome and gene annotation set. Sequence alignments were compressed and sorted into binary alignment map files using samtools version 1.3. Mapped reads for genomic features were counted using Subread featureCounts version 1.6.2 using the parameters -s 2 –g gene_id–t exon–Q 60; the annotation file specified with —a was the NCBI GRCh38 reference from Illumina iGenomes ,31,32.p-value  < 0.05 and with an absolute fold change ≥ 1.3 were considered differentially expressed. To visualize how differentially expressed genes (DEGs) could separate control and IA samples, we performed hierarchical clustering  on scaled log-transformed data and visualized the results in a heatmap. We also performed k-means clustering using 1-pearson correlation metric, k = 2, and 1000 iterations .For differential expression analysis raw counts were normalized as transcripts per million (TPM). Protein coding genes with expression in >50% of all samples were considered for differential expression analysis using an F-test. Those with a John Storey False Discovery Rate (FDR) corrected https://cibersort.stanford.edu/, accessed on 22 April 2021) [n = 9239). A Student’s t-test was used to evaluate if there were any significant differences in predicted cell populations between the IA and control groups .Cell composition analysis was performed using the open-access CIBERSORT application  . Analysihttps://biit.cs.ut.ee/gprofiler/gost, accessed on 22 April 2021) in g:Profiler program [p-values, were summarized and visualized as networks in the REVIGO  tool [Based on our differential expression analysis, we studied ontological enrichment in our significantly differentially expressed genes using the g:GOSt tool  tool  using a https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis, accessed on 22 April 2021) [p-scores > 20 were considered significant. We also assessed predicted upstream regulators in IPA. Biomolecules  with a Benjamini–Hochberg FDR-corrected p-value < 0.05 were considered regardless of z-score.Additional analyses were performed in Ingenuity Pathway Analysis  . Here, ITo determine if differentially expressed genes could also separate patients with IAs from controls in an independent cohort, we evaluated their expression in the validation cohort that consisted of 25% of all samples. For comparison, we calculated their fold-change in this new cohort. Principal component analysis using the prcomp package in R was used to determine if the genes could delineate IA from controls in both cohorts. To further explore their diagnostic potential, we performed supervised machine learning on the TPM data using the MATLAB Statistics and Machine Learning Toolbox . In the discovery dataset, we trained a Subspace Discriminant Ensemble model with four-fold cross-validation to avoid overfitting. This model was then tested in the validation dataset. In training and testing, we calculated the model’s accuracy, sensitivity, and specificity.p-values from the Wald test (α = 0.05). A “very strong” correlation was defined by 1.0 ≥ |PCC| ≥ 0.80, 0.79 ≥ |PCC| ≥ 0.60 defined “strong” correlation, 0.59 ≥ |PCC| ≥ 0.40 defined “moderate” correlation, 0.39 ≥ |PCC| ≥ 0.20 defined “weak” correlation, and |PCC| < 0.19 defined a “very weak” or no correlation [As aberrant RNA expression in circulating PBMCs may be aggravated in cases of larger or more developed IAs, we explored the relationship between expression of DEGs and IA risk. We assessed risk in two ways: (A) By measuring IA size  and (B) By calculating 5-year rupture risk % according to the International Study of Unruptured Intracranial Aneurysms (ISUIA) study . To idenWe studied the gene expression profiles from a total of 52 peripheral blood samples  that met our inclusion and quality criteria. As shown in q > 0.05). On average, monocytes represent the majority (29%) followed by CD4+ T cells (28%), NK cells (18%), CD8+ T cells (12%), B cells (4%), Tregs (3%), neutrophils (3%—likely a contaminant from processing), mast cells (1%), and macrophages (1%)  see .q < 0.05 and an absolute fold-change ≥ 1.3) between the two groups of the discovery cohort, reported in The volcano plot in To understand the biological implication of differential PBMC RNA expression, we performed several bioinformatics analyses. We performed detailed gene ontology term enrichment analysis using g:Profiler, then reduced and visualized GO terms using REVIGO. Networks of significant GO terms for the significant up- and down-regulated genes are shown in p-score > 20) relating to the significant genes. The first network . Network 2 . See All 54 significantly differentially expressed genes were also analyzed using IPA. As demonstrated in  network A had a petwork 2 B had a petwork 3 C also han = 13 patients (see n = 6) had aneurysms ranging in size from 3 to 10 mm (average size = 6.4 mm) and included one individual with multiple aneurysms. TPM levels from RNA sequencing data for the 54 genes of interest were used to visualize how these transcripts could distinguish the IA group from the control group. PCA using these genes in both the discovery and validation cohort are shown in To determine whether expression of the IA-associated signature could separate patients with IAs from controls in an independent cohort, we performed a small replication study in the validation cohort of MKRN3—PHGDH—TIMD4—TRIM7—We used Pearson correlation analysis to explore the relationship between the expression of the 54 significant genes and IA risk, as measured by aneurysm size and 5-year rupture risk % (from ISUIA). As shown in It is widely known that the natural history of IA is accompanied by progressive inflammatory responses in the vascular wall ,12,13,14The role of mononuclear cells has been widely studied in IA . PBMCs aConsidering the critical role of circulating PBMCs in IA inflammation and that they are actively recruited to the aneurysm wall from the blood, we hypothesized that expression patterns in PBMCs may be altered in patients harboring IAs compared to their IA-free counterparts. Transcriptome profiling by next-generation RNA sequencing identified a signature of 54 differentially expressed genes with an absolute fold change of ≥1.3 . The majority of these genes maintained the same expression pattern in an independent validation cohort of patients and could delineate patients with IA from controls based on our PCA and machine learning analysis (Ensemble classifier). To our knowledge, only one other study has investigated circulating gene expression patterns of IA in PBMCs. Sabatino et al. collected PBMCs from a cohort of 15 unruptured IAs, 15 ruptured IAs, and 15 control patients and performed gene-expression oligo-microarrays on isolated PBMC RNA . In all,HSPA2), as well as differential expression of many genes involved in intracellular signaling, such as g-protein coupled receptors , and plasma membrane ion channels . Other IA-associated genes we identified have also been found to be differentially expressed in various blood components of IA patients. ANKRD22, which was increased in our study, was also increased in whole blood expression profiles as reported by Zhao et al. [CCR8, G0S2, SDC3  and UTS2 (decreased) were also seen in previous work from our group, in which we investigated expression signatures in circulating neutrophils as well as whole blood from patients with IA [We designed the current study to avoid previous pitfalls and ensure greater accuracy of results. To do this, we confirmed diagnoses on DSA imaging, cohort-controlled our datasets, and implemented a small replication study. These measures helped to increase the likelihood that the discovered signature is truly associated with the presence of unruptured IA. Additionally, we used next-generation RNA sequencing. Unlike microarrays used in past studies, RNA sequencing offers a larger dynamic range, facilitating detection of expression differences in low-abundance transcripts, and avoids predetermined probes, allowing for examination of novel RNAs  ,55. Baseo et al. . Express with IA ,7,9.HLA-DQB2 (a major histocompatibility complex) and CCR8. CCR8 is related to inflammatory signaling, as it is in the beta chemokine receptor family and has increased expression in M1 pro-inflammatory macrophages (compared to M2 macrophages), which have also been shown to be more prevalent in IAs and may contribute more to pathologic remodeling during IA [G0S2 [TIMD4 in IA PBMCs also indicates the activation of peripheral blood monocytes and T cells, as it has been a marker of recruited macrophages and macrophage polarization and is involved in T cell proliferation [In this study, we detected many other significant genes, which broadly indicated peripheral activation of PBMCs during IA. Our bioinformatics analyses show that PBMCs from IA patients reflect a complex reaction to intravascular perturbations that is associated with increased cell activation, cell signaling, adhesion, and regulation of extracellular matrix. The activation of circulating immune cells, likely monocytes and T cells, is most reflected in enriched “response to stimulus” and “cell adhesion” ontologies. These are related to increased expression of uring IA ,51. ActiIA [G0S2 . As demoferation ,59,60.ADAMTS17, a member of the ADAMTS family that is involved in proteoglycan cleavage along with MMPs [PTGS2 or COX2. COX2 is an inducible enzyme responsible for prostaglandin production, and as such, plays an essential role in inflammatory pathways [COX2, and other important genes , is coordinated through TNF (in addition to NF-kB), as evidenced by our upstream analysis in IPA. TNFα is a critical part of the immune system and has been demonstrated to be a key mediator of inflammatory cell infiltrates in IA disease [In addition to activation, gene ontology term enrichment analysis revealed increased structural development . In our network analysis, this was conferred through several ADAMTS nodes and their connections to collagens and TGFB (a key inflammatory regulator in IA). Indeed ith MMPs ,62, was pathways ,64. Its pathways ,66. One pathways ,68,69. W disease ,70. We sMKRN3 and TRIM7, and IA size and 5-year rupture risk (ISUIA). While the role of MKRN3 is relatively unknown, TRIM7 has been shown to promote TLR4-mediated signaling activation, which via NF-kB, leads to the production of proinflammatory cytokines and type I interferon [PHGDH and TIMD4, and IA size and rupture risk. PHGDH encodes an enzyme that is involved in L-serine synthesis, and has been shown to have decreased expression in aneurysm tissue [TIMD4, the correlation analysis may also indicate decreased T cell proliferation in patients with larger IAs. This may be akin to findings by Korostynski et al., who found a decrease in CD4+ lymphocytes in the blood of patients with ruptured IAs [Given the potential role of NF-kB and TNF (two key inflammatory mediators in IA tissue ) in reguterferon . This com tissue . The negured IAs . Larger,In summary, transcriptome profiling of PBMCs in patients with IA revealed a signature of 54 significantly differentially expressed genes. These genes are likely to be related to biological processes known to be enriched in IAs, such as NF-kB signaling and TNF activity. Because of this relationship to the disease, we posit that this signature, upon future rigorous validation, may be a potential biomarker for IA presence. Previous work in this field has identified circulating protein markers, such as VEGF  or MCP-1This preliminary study has several limitations. First, our study had a relatively small sample size. However, demonstrating separation of IAs from controls in an independent cohort gives confidence in the identified signature. Future studies in larger cohorts will increase the statistical power in identifying differentially expressed genes. Second, the study subjects were recruited from patients receiving cerebral imaging (which was necessary to confirm the presence or absence of IA) at a single center. This may have introduced a potential selection bias. Future studies in broader, randomized populations from multiple centers are needed. Third, despite careful patient selection to ensure matched cohorts, there were some differences  in the rates of patients’ characteristics, such as smoking and osteoarthritis, which could affect the data. Future efforts in larger datasets should be made to normalize results based on patient demographics and comorbidities. Lastly, there is a possibility that the expression signature could be influenced by other conditions not accounted for. Additional studies should investigate the specificity of the differential expression profiles to IA.q-value < 0.05. These genes also separated patients with IAs from controls in a small validation cohort. Bioinformatics analyses demonstrated enrichment of structural regulation processes, intracellular signaling function and regulation of ion transport. IPA analysis showed that these processes were likely coordinated through NF-kB, cytokine signaling, growth factors, and TNF activity. Our machine learning analysis highlights the exciting potential to develop circulating RNA expression-based diagnostics for IA in future, larger studies.In this preliminary study, we performed transcriptome profiling on PBMCs from patients with and without IAs and identified a signature of 54 differentially expressed genes with an absolute fold-change of ≥1.3 and


Background: Antenatal depression (AD) is a major public health issue worldwide and lacks objective laboratory-based tests to support its diagnosis. Recently, small metabolic molecules have been found to play a vital role in interpreting the pathogenesis of AD. Thus, non-target metabolomics was conducted in serum.Methods: Liquid chromatography—tandem mass spectrometry—based metabolomics platforms were used to conduct serum metabolic profiling of AD and non-antenatal depression (NAD). Orthogonal partial least squares discriminant analysis, the non-parametric Mann–Whitney U test, and Benjamini–Hochberg correction were used to identify the differential metabolites between AD and NAD groups; Spearman's correlation between the key differential metabolites and Edinburgh Postnatal Depression Scale (EPDS) and the stepwise logistic regression analysis was used to identify potential biomarkers.Results: In total, 79 significant differential metabolites between AD and NAD were identified. These metabolites mainly influence amino acid metabolism and glycerophospholipid metabolism. Then, PC (16:0/16:0) and betaine were significantly positively correlated with EPDS. The simplified biomarker panel consisting of these three metabolites  has excellent diagnostic performance  in discriminating AD and NAD.Conclusion: The results suggested that betaine, PC (16:0/16:0), and succinic acid were potential biomarker panels, which significantly correlated with depression; and it could make for developing an objective method in future to diagnose AD. Antenatal depression (AD), defined as depression during pregnancy up to the point of birth, is one of the most widespread psychiatric disorders and produces harmful effect on the mother and the infant's health , 2. SystAs it is well-known, depression disorder is greatly influenced by genetics and environmental factors, as well as their interactions , 10. AD Metabolomics, a promising tool in non-invasive biomarkers for diagnosis, has been widely used to identify global or targeted endogenous metabolites by detecting various biosamples, such as urine and plasma , 21. CurThus, a serum metabonomic method in the diagnosis of AD was evaluated. LC-MS–based serum metabolomic platforms were conducted to distinguish metabolic profiling of AD and non-antenatal depression (NAD). Besides, orthogonal partial least squares discriminant analysis was used to identify the differential metabolites between the two groups; Spearman's correlation between key differential metabolites and Edinburgh Postnatal Depression Scale (EPDS), and the stepwise logistic regression analysis was used to identify potential biomarkers.2; (5) complete clinical data; and (6) no history of mental or cognitive illness before pregnancy. The exclusion criteria were as follows: (1) educational level <9 years; (2) history of psychoactive substance abuse; (3) with severe systemic diseases such as metabolic encephalopathy and liver or kidney disease; and (4) complications associated with pregnancy.The protocols of this research were reviewed and approved by the Medical Ethics Committee of The Affiliated Hospital of Guizhou Medical University. All participants were asked to sign informed consent before sample collection; also, all procedures were conducted according to the principles expressed in the Declaration of Helsinki. In present study, 60 subjects were recruited from the psychiatry department of the Affiliated Hospital of Guizhou Medical University from September 2015 to September 2016. All singleton-pregnancy subjects were screened by licensed obstetricians and psychiatrists. The inclusion criteria were as follows: (1) late pregnancy ; (2) parity ≤ 4; (3) between the ages of 22 and 40 years; (4) body mass index (BMI) at prepregnancy >18.5 and <30 kg/mThe diagnosis of perinatal depression (AD) was implemented based on a renowned approach as described in the previous studies , 26. AllThe fasting blood was collected from the elbow vein of subjects in the morning using 5-mL Vacutainer tubes. Then, the blood was allowed to static 30 min at room temperature and followed by centrifugation at 3,000 revolutions/min (rpm) for 15 min. Each aliquot (150 μL) of the plasma sample was stored at −80°C until further analyses. The sample extraction protocol was based on previously published literature . BrieflyThe Vanquish™ UHPLC system  with a Waters ACQUITY UPLC BEH Amide column  was used to separate the metabolites, respectively. The temperature of column and autosampler was set at 25°C and 4°C, respectively, and the volume of injection was 3 μL The mobile phase (A) was prepared by dissolving 25 mmol/L ammonium acetate and ammonia hydroxide in water and adjusted the pH of solution to 9.75, and the mobile phase (B) consisted of 100% acetonitrile. The analysis was carried out with elution gradient as follows: 0–0.5 min, 5% A; 0.5–7.0 min, 5–35% B; 7.0–8.0 min,35–60% A; 8.0–9.0 min, 60% A; 9.0–9.1 min, 60–5% A; 9.1–12.0 min, 5% A .The Q Exactive HFX mass spectrometer  system and MS/MS data acquisition control were performed by acquisition software . Specification of the ESI source was set as follows : (1) theU test was conducted to analyze the metabolites, and multiple test corrections using the Benjamini–Hochberg correction were applied to valuate statistical significance. The metabolites with VIP values >1.0 and Q value < 0.05 were considered to be statistically significant, whereas variables that were not significantly changed were discarded , which uses the high-quality Kyoto Encyclopedia of Genes and Genomes metabolic pathways as the backend knowledge base . Heatmap and Spearman's rank correlation analysis of key differential metabolites were conducted using RStudio. Then, the stepwise binary logistic regression analysis and receiver operating characteristic (ROC) curve analysis were performed to explore biomarkers for distinguishing NAD from AD. The area under the curve (AUC) was used to assess the accuracy of diagnostic accuracy, such as 0.8 < AUC < 0.9 as good and 0.9 < AUC ≤ 1.0 as excellent  or RStudio version 1.2.1335-2009-2019  software. Data were assessed for normality of distribution using the Shapiro–Wilk test first. The two-tailed Student p > 0.05) in age, BMI of pre-pregnancy and pregnancy, gestational weeks, or education level between the two groups.The clinical and anthropometric characteristics of all the participants included in the study are summarized in LC-MS/MS metabolomic platform was used to assess the contributions of serum metabolome. After data normalization, the OPLS-DA model revealed that the AD and NAD patients could be significantly separated with little overlap in the negative mode  against the corresponding log2 fold change (AD/NAD) (x axis), and the differential metabolites were screened according to the set criteria (VIP > 1 and Q < 0.05). The volcano plot represents the significant variables in the discrimination between AD and NAD groups in the negative  and amino acid metabolism, such as glycine, serine, and threonine metabolism (p = 0.0001) and Alanine, aspartate, and glutamate metabolism (p = 0.0334).All the differential metabolites were mapped into their biochemical pathways through metabolic enrichment and pathway analysis based on the database MetaboAnalyst 5.0, the overview of pathway enrichment and analysis is shown in p < 0.05 , includir = 0.537, p < 0.001) and betaine  were significantly positive correlated with EPDS, and l-serine  was not  and succinic acid] could yield an AUC of 0.960  . These rTo our knowledge, this is the first study to use LC-MS/MS platform to evaluate serum metabolites in late-pregnancy women with and without depression symptoms. In the overall analysis, 79 metabolites were significant different between AD and NAD, which were related to amino acids and lipids (mainly about fatty acid and glycerophosphocholines). Specifically, we found that most of the key metabolites were related to “amino acid metabolism” including “glycine, serine, and threonine metabolism,” “alanine, aspartate and glutamate metabolism,” and “glycerophospholipid metabolism.” Furthermore, correlation analysis revealed that PC (16:0/16:0) and betaine were associated with higher AD risk. Finally, a potential biomarker panel consisting of three serum metabolite biomarkers  was identified. This panel could discriminate pregnant women with AD from those with NAD with AUC of 0.960.Depression is a heterogeneous and multifactorial disorder, while the molecular mechanisms that underlie depression remain unclear. Recently, a growing body of evidence suggests that lipid metabolism plays a vital role in the pathological of depression , 37–39. Depression, a serious mental illness, is influenced by genes and the environment. It has reported that the environmental factors regulated the gene expression and function of neuronal membrane via epigenetic mechanisms . CholineN-methyl-d-aspartate receptors (NMDARs) (l-glutamic acid (Glu), succinic acid, and citric acid decreased in the serum of pregnant women with depression, which was related to alanine, aspartate, and glutamate metabolism pathway. In line with previous studies, Glu decreased in the dorsolateral prefrontal cortex of individuals with depression, as well as in the anterior cingulate cortex  , such as(NMDARs) . In thise cortex . In addie cortex . Glu alse cortex . It is te cortex . TherefoSome limitations need to be addressed. First, relatively small sample size and late-pregnancy subjects were enrolled in this study; future studies with large-scale samples are still needed to validate our study. Second, all subjects were from the same location and might share the same dietary habits, which may restrict the generalization of the findings. Third, only serum metabolites were studied, and further studies should collect other biological samples from the same subjects. Fourth, the number of differential metabolites might be not enough to obtain the robust results of pathway analysis and enrichment analysis; thus, future studies are still needed to validate and support these results. Therefore, these findings shed new light to further elucidate the molecular mechanism of depression. And our preliminary investigation found that studies on potential biomarkers for AD and key metabolic pathways are needed for further validation.In summary, 79 significant differential metabolites between AD and NAD were identified by LC-MS/MS. We also found that these metabolites mainly influenced “amino acids metabolism” and “glycerophospholipid metabolism.” Meanwhile, potential serum diagnostic metabolite panels  clearly discriminated AD from NAD with excellent accuracy. These findings may aid in uncovering the molecular pathogenesis of AD and then prompting the development of diagnostic and prognostic tests for the disorder.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the affiliated hospital, Guizhou Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.TZ: study concept and design. TT, XG, and XZ: performed the experiments. TT: experimental technical guidance. QM: data analysis, manuscripts drafting, and revised. All authors reviewed and approved the manuscript before its submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To systematically review the literature evaluating clinical utility of imaging metrics derived from baseline fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) for prediction of progression-free (PFS) and overall survival (OS) in patients with classical Hodgkin lymphoma (HL) and diffuse large B cell lymphoma (DLBCL).A search of MEDLINE/PubMed, Web of Science, Cochrane, Scopus and clinicaltrials.gov databases was undertaken for articles evaluating PET/CT imaging metrics as outcome predictors in HL and DLBCL. PRISMA guidelines were followed. Risk of bias was assessed using the Quality in Prognosis Studies (QUIPS) tool.Forty-one articles were included . Significant predictive ability was reported in 5/20 DLBCL studies assessing SUVmax , 17/19 assessing metabolic tumour volume (MTV)  and 10/13 assessing total lesion glycolysis (TLG) . Significant predictive ability was reported in 1/4 HL studies assessing SUVmax (HR not reported), 6/8 assessing MTV  and 2/3 assessing TLG (HR not reported). There are 7/41 studies assessing the use of radiomics ; 5/41 studies had internal validation and 2/41 included external validation. All studies had overall moderate or high risk of bias.Most studies are retrospective, underpowered, heterogenous in their methodology and lack external validation of described models. Further work in protocol harmonisation, automated segmentation techniques and optimum performance cut-off is required to develop robust methodologies amenable for clinical utility.The online version contains supplementary material available at 10.1007/s00259-021-05233-2. Lymphoma is a haematopoietic malignancy, which can be broadly categorised into Hodgkin and non-Hodgkin disease. Hodgkin lymphoma (HL) accounts for approximately 10% of all newly diagnosed cases, and its hallmark is the presence of Hodgkin and Reed–Sternberg (HRS) cells . Non-HodThere are several pretreatment clinical prognostic tools developed to stratify both DLBCL and HL. In 1993, Shipp et al. introduced the international prognostic index (IPI) for predicting overall survival in DLBCL patients based on a retrospective study of 2031 patients treated with CHOP. The IPI has been further refined with an age-adjusted version (aa-IPI), a revised version developed following the use of R-CHOP (R-IPI), and a version based on the National Comprehensive Cancer Network database (NCCN-IPI). HL disease can be split into early (stage I and II) or advanced (stage III or stage IV) with early being split into favourable or unfavourable depending on one of the many scoring systems including, but not limited to, the German Hodgkin Study Group (GHSG), European Organisation of Research and Treatment of Cancer (EORTC), Groupe d’Etudes des Lymphomes de l’Adulte (GELA), National Cancer Institute (NCI) or National Comprehensive Cancer Network 2010 (NCCN 2010) scores. However, given the variation in the prognostic groups derived from the different scoring systems, further information obtained from imaging may improve prognostication.2-deoxy-2-[Fluorine-18]fluoro-D-glucose (FDG) positron emission tomography/computed tomography (PET/CT) is widely used for staging and response assessment in HL and NHL . ResponsA variety of imaging-derived quantitative parameters have been reported in the literature with potential utility for predicting prognosis or treatment outcome. These metrics range from those based on tumour volume to metabolic features, including shape and texture. At present, none have been translated into routine clinical practice. The purpose of this study was to perform a systematic review of the literature reporting the use of quantitative imaging parameters derived from pretreatment FDG PET/CT for prediction of treatment outcome for HL and DLBCL. Due to the varied nature of NHL, DLBCL was chosen as it is the most common subtype of NHL.clinicaltrials.gov databases was performed for articles on PET/CT imaging parameters in lymphoma treatment assessment. The search strategy included three primary operator criteria linked with the “AND” function. The first criteria consisted of “lymphoma”, the second of “PET” or “positron emission tomography”, and the third of “outcome”, “prognosis”, “prediction”, “parameter”, “radiomics”, “machine learning”, “deep learning” or “artificial intelligence”. Case studies, articles not published in English, phantom studies, studies not assessing treatment outcomes using baseline imaging in HL or DLCBL, studies assessing primary anatomical presentations of lymphoma or HIV-related lymphoma, mixed pathology studies and studies assessing novel treatments were excluded. After duplications were excluded, studies were screened for eligibility based on the title, abstract and subsequently on full text. The references of the articles included in the systematic review were manually reviewed to identify further publications which met the inclusion criteria. The results were stored in bibliographic management software. Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) criteria were adhered to [A search of MEDLINE/PubMed, Web of Science, Cochrane, Scopus and hered to .The Quality in Prognosis Studies (QUIPS) tool was used to evaluate validity and bias which considers six areas: inclusion, attrition, prognostic factor measurement, confounders, outcome measurement, and analysis and reporting . PromptiResults are current to July 2020. The database search strings yielded 2717 results after duplicates were excluded. Following screening and assessment of eligibility, 41 articles meeting the study inclusion criteria were included. Figure No studies showed low risk of bias in all six domains  guidelines with their scanning protocol; 10/41 did not take into consideration important co-founders such as different treatment regimes, stage, prognostic scores or histology. Only six studies defined the method for calculation of SUV, and 7 studies used a validation cohort to test the predictive models  and reporting bias is present because a number of studies which did not report any significance did not provide the results required to calculate a HR.The majority of studies assessing the use of baseline SUVmax in DLBCL report no significant ability to predict progression-free survival (PFS) or overall survival (OS) Table . Forest b)HLOf the studies which showed a prognostic ability for SUVmax, Gallicchio et al. reported this was the only imaging parameter able to predict PFS when compared to TLG and MTV in a small study of 52 DLBCL patients (26 early and 26 advanced stage) with a higher SUVmax associated with a longer PFS, the hazard ratio (HR) was 0.13 (0.04–0.46) . A study, who also found no significant ability of SUVmax to predict PFS or OS in 258 stage I and II patients. Angelopoulou et al. reported that SUVmax was a significant predictor of 5-year PFS in a study of 162 patients with a split of stages  [Five studies have assessed the use of SUVmax as a predictive parameter in HL patients with only one reporting significance Table . The larIV = 86) . The cohIV = 86) . They foFactors affecting SUV such as scanner spatial resolution, image acquisition and PET reconstruction parameters combined with a relatively small number of events, variation in the number of early and advanced patients, differences in treatment and definition of PFS all influence the results , 52. ThiDLBCLThe potential utility of baseline MTV and TLG for predicting PFS and OS in patients with DLCBL has been reported in multiple studies Table  and 5. H3 had significantly better PFS and OS; 89.8 versus 55.6%, and 93.2 versus 58.0%, respectively [3 and BMI had worse PFS and OS survival compared to those with a smaller MTV and BMI [3 and a Dmax <58 cm had a 4-year PFS of 94% and OS of 97%, the intermediate group with either an MTV >394cm3 or a Dmax >58 cm had a 4-year PFS of 73% and OS of 88% and the high-risk group with a MTV >394cm3 and a Dmax >58 cm had a 4-year PFS of 50% and OS of 53%.One of the largest studies by Song et al. evaluated 169 patients with DLBCL  treated with R-CHOP . Patientectively . MTV was and BMI . Again,  and BMI . The spap = 0.012). A possible explanation for the discrepancy between the prognostic ability of MTV and TLG in this study may be related to the correlation between MTV and TLG, confounded by relatively small sample sizes. Kim et al. evaluated TLG calculated using different MTVs derived using 25, 50 and 75% SUVmax thresholds in a mixed cohort (n = 140) of early and advanced stage DLBCL patients being treated with R-CHOP [3 region of interest (ROI) within the right lobe of the liver (PERCIST) using the Hermes software. They found a strong agreement between all four methods, with the lowest intraclass coefficient being between PERCIST and 41% SUVmax thresholds being 0.86. They also reported similar receiver operator curves (ROC) between the four methods with the area under the curve (AUC) ranging from 0.74 to 0.76 for PFS, and 0.71 to 0.75 for OS. All four methods were significant predictors of PFS and OS. However, as stated in the paper, no method is likely to apply to all patients generally. Large heterogeneous masses are likely to be undersized with percentage thresholds, low uptake lesions may be missed using a standard threshold method and disease involving the liver may impede its use as the background value. This may have a more significant impact when further metrics are introduced, such as those based on texture when the size of the contour can also influence the reported values. The segmentation technique of choice also needs to be easily replicated. Recently, Capobianco et al. assessed the use of artificial intelligence (AI) using a convolutional neural network (CNN) to segment the MTV [Zhou et al. reported that although high baseline MTV and TLG were associated with poorer prognosis, only TLG was an independent predictor of PFS and OS in a study of 91 patients . In thish R-CHOP . They foh R-CHOP . The fou the MTV . They foI2 = 74% for PFS and I2 = 70% for OS.Fewer studies have investigated the predictive ability of MTV and TLG in HL patients than in DLBCL Table  and 7. TSimilar to DLBCL, clinical implementation of MTV and TLG in HL depends on reaching a consensus regarding segmentation methodology, each giving different variations in the volumes measured and will be facilitated by an automated process. However, variation in treatment is likely also to play an impact, and this aspect needs assessing in larger multi-centre studies.Textural analysis or radiomics relates to transformation of images into mineable high-dimensional data permitting invisible feature extraction, analysis and modelling for non-invasive phenotyping and outcome prediction . Radiomin = 105) and validation sets (n = 27) and found that Long-Zone High-Grey Level Emphasis (LZHGE) was the only independent predictor when analysed with IPI and MTV. On the validation set, it was found that a high LZHGE > 1,264,925.92 was associated with a 2-year PFS of 60% whereas patients with a low LZGHE had a PFS of 94.1%. The study has some limitations as only the largest area of disease was analysed, a breakdown of disease stage was not presented and 14 patients did not have standard (R-CHOP) therapy. Another study by Aide et al. investigated the diagnostic and prognostic value of axial skeletal textural features derived from PET/CT in patients with DLBCL in a retrospective cohort of 82 patients [Aide et al. studied the use of PET/CT-derived textural features, clinical and imaging parameters to predict 2-year PFS in DLBCL patients . They sppatients . The CT Lue et al. investigated the use of 11 first-order, 39 higher-order features and 400 wavelet features for predicting PFS and OS in 42 HL patients  with 21 events within the cohort  . They fon = 12). Five features were selected as the most predictive . InformationMeasureCorr1 and InformationMeasureCorr2 are the first and second measures of theoretic correlation and Inverse-Variance is weighting of random variables to minimise variance. By combining these features, the AUC for predicting relapse for patients with mediastinal disease was 0.95. This outperformed TLG and MTV. This work highlights the potential for using AI-methods in lymphoma assessment. However, the study is limited to HL with mediastinal involvement with again small numbers of events.Decazes et al. retrospectively studied PET/CT scans of 215 DLBCL patients to assess the utility of total tumour surface (TTS) and tumour volume surface ratio (TVSR) as predictive biomarkers , TVSR ben = 86) and validation cohorts (n = 64) treated at two centres [P < 0.001 and 5-year PFS, 84.1 vs. 43.6 vs. 27.0%, P < 0.001 respectively.Senjo et al. demonstrated that a high metabolic heterogeneity (MH) was a predictor of 5-year PFS and OS in DLBCL across both training ESM 2(DOCX 55 kb)


Crangonyx forbesi) and four groundwater amphipods  from eastern North America using a shotgun sequencing approach on an Illumina HiSeq 4000 . All five mitochondrial genomes encoded 13 protein-coding genes, 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs) representative of subphylum Crustacea. Although the four groundwater species exhibited gene orders nearly identical to the ancestral pancrustacean gene order, the spring-dwelling species, C. forbesi, possessed a transposition of the trnH–nad4–nad4l loci downstream after nad6–cytb–trnS2. Moreover, a long nad5 locus, longer rrnL, and rrnS loci, and unconventional start codons distinguished C. forbesi from the four groundwater amphipods. Overall, our five amphipod mitogenomes add to the increasing publicly available mitogenome resources for amphipods that are not only valuable for studying the evolutionary relationships of this diverse group of crustaceans but for exploring the evolution of mitochondrial genomes in general.We sequenced the mitochondrial genomes of one spring-dwelling ( Bactrurus, Crangonyx, and Stygobromus  , in May 2015. Specimens of Bactrurus brachycaudus were collected from Fogelpole Cave  , in October 2014. Specimens of Stygobromus allegheniensis were collected from Caskey Spring  , in September 2013. Specimens of Crangonyx forbesi were collected from a unidentified spring , in 2013. All specimens were preserved in 100% ethanol in the field. Specimens and DNA extracts are maintained in the CaveBio collection at The University of Alabama in Huntsville. Whole genomic DNA was isolated using the Qiagen DNA Easy Blood and Tissue kit and libraries were prepared using the Illumina TruSeq DNA Library Prep Kit . Libraries were then paired-end sequenced (2 × 150 bp) on an Illumina HiSeq 4000 platform at the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley (supported by NIH S10 OD018174 Instrumentation Grant).Specimens of C. forbesi mitogenome. The secondary structures of tRNAs were inferred using MITFI , Stygobromus tenuis potomacus , Stygobromus allegheniensis , Bactrurus brachycaudus , and Crangonyx forbesi . All mitogenomes contained 13 protein-coding genes, 22 tRNA genes, one small ribosomal RNA (rrnS) gene, and one large ribosomal RNA (rrnL) gene, and a non-coding control region representative of the Kingdom Animalia. In all mitogenomes, the heavy strand encoded a total of 23 genes, whereas the light strand encoded 14 genes. Similar to the ancestral gene order of pancrustaceans  than B. brachycaudus, S. pizzinii, or S. tenuis potomacus, which all possessed just eight intergenic spacers. These intergenic spacers were found primarily between protein-coding genes, but at times also between tRNA genes. The high number of spacers in C. forbesi and S. allegheniensis could be a result of gene rearrangement . This length difference is because of additional nucleotides found at the 3′ end of the nad5 gene. Contrastingly, the nad2 gene of S. allegheniensis was 895 bp in length and was shorter than nad2 in other species (943–1023 bp). In addition, the nad6 gene of B. brachycaudus was 531 bp in length and was longer than in other species (486–507 bp), while the cytb gene was 1098 bp in length and was shorter than in other compared species (1117–1140 bp). The atp6 gene of S. tenuis potomacus, S. pizzinii, and S. allegheniensis overlapped with the adjacent atp8 gene by 41 bp, whereas no overlap was observed in B. brachycaudus and C. forbesi. An overlap between the atp8 and atp6 genes has been reported in other Stygobromus species including S. tenuis and S. indentatus  and both loci were flanked by tRNAs trnL1 and trnI. The length of the rrnL gene in the other crangonyctid species (1034–1037 bp) was similar to that of other amphipod species.Alignment of the large ribosomal RNA (rrnL) gene of all five crangonyctid amphipods revealed the presence of a long sequence (52 bp) overhang on the 5′ end of the rrnL gene of C. forbesi. The 3′ end of the rrnS gene in all crangonyctid mitogenomes was followed by a continuous stretch of thymines, which was identified as the 5′ end of the non-coding control region. However, the rrnS gene in C. forbesi was immediately followed by tRNA trnl. The control region of C. forbesi contained a transposition and was located downstream of the nad1 gene.The small ribosomal RNA (rrnS) gene of all five amphipod species had a relatively conserved 3′ end. Analogous to the rrnL gene, a long sequence (47 bp) overhang was observed on the 5′ end of the rrnS gene in S. tenuis potomacus mitogenomes revealed few differences. Their gene order and AT content (69.1%) were identical, and both had equal numbers of intergenic spacers. The unconventional start codon GTG for the cox2 locus found in the S. tenuis potomacus (MN175621) mitogenome we sequenced was not observed in the S. tenuis potomacus sequenced previously  used the incomplete stop codon T—while the conventional stop codon TAA was used in S. tenuis potomacus (MN175621). In addition, the stop codon for nad3 locus differed in their 3rd position (TAA versus TAG) between the two mitogenomes. Another difference observed was in lengths of the non-coding control region and nad3 locus found. The control region of S. tenuis potomacus (KU869712) was 773 bp in length and was substantially larger than in S. tenuis potomacus . In addition, the nad3 locus of S. tenuis potomacus (KU869712) was 381 bp in length and larger than in S. tenuis potomacus . Apart from these differences, the location, structure, and length of transfer and ribosomal RNAs were nearly identical between the two mitogenomes.A comparison between the two Bactrurus, Crangonyx, and Stygobromus) form a well-supported clade. However, the genus Stygobromus was not monophyletic, as B. brachycaudus and C. forbesi were nested within Stygobromus. Crangonyx forbesi was sister to all other crangonyctids, although support for this relationship was lower. The paraphyly of Stygobromus also was uncovered in a phylogenetic analysis of the cox1 locus (Niemiller et al. Bayesian phylogenetic inference of the 13 protein-coding gene alignment  yielded S. pizzinii, S. allegheniensis, S. tenuis potomacus, and B. brachycaudus – as well as the complete mitogenome of a spring-dwelling amphipod C. forbesi to the growing list of publicly available amphipod mitogenomes. Although all five new mitogenomes exhibited the complete set of 37 genes commonly observed in other amphipods, C. forbesi displayed several unique gene arrangements, including the transposition of genes trnH–nad4–nad4l downstream after nad6–cytb–trnS2. This particular gene arrangement deviates significantly from the ancestral pancrustacean gene order and has not been detected in other amphipod species to date. Phylogenetic analysis supports the monophyly of Crangonyctidae but suggests that the genus Stygobromus is not a monophyletic group. In general, our contribution of these five additional crangonyctid mitogenomes will be highly valuable for inferring the phylogenetic relationships, biogeography, and trait evolution of amphipods and investigating mitogenome evolution.In this study, we have added the complete mitogenomes of four groundwater amphipods –