Prochlorococcus were detected throughout the entire water column. We proposed that vertical sinking, heterotrophic metabolism, and/or the transition to resting stage of phytoplankton might contribute to the presence of phytoplankton in the hadal zone. This study provided insight into the PN community in the Mariana Trench, implied the significance of phytoplankton in exporting organic matters from the euphotic to the hadal zone, and also hinted the possible existence of some undetermined energy metabolism  of phytoplankton making themselves adapt and survive in the hadal environment.As photoautotrophs, phytoplankton are generally present in the euphotic zone of the ocean, however, recently healthy phytoplankton cells were found to be also ubiquitous in the dark deep sea, i.e., at water depths between 2000 and 4000 m. The distributions of phytoplankton communities in much deeper waters, such as the hadal zone, are unclear. In this study, the vertical distribution of the pico- and nano-phytoplankton (PN) communities from the surface to 8320 m, including the epipelagic, mesopelagic, bathypelagic, and hadal zones, were investigated via both 18S and p23S rRNA gene analysis in the Challenger Deep of the Mariana Trench. The results showed that Dinoflagellata, Chrysophyceae, Haptophyta, Chlorophyta, Prochloraceae, Pseudanabaenaceae, Synechococcaceae, and Eustigmatophyceae, etc., were the predominant PN in the Mariana Trench. Redundancy analyses revealed that depth, followed by temperature, was the most important environmental factors correlated with vertical distribution of PN community. In the hadal zone, the PN community structure was considerably different from those in the shallower zones. Some PN communities, e.g., Eustigmatophyceae and Chrysophyceae, which have the heterotrophic characteristics, were sparse in shallower waters, while they were identified with high relative abundance  at the depth of 8320 m. However, the dinoflagellates and Prochloraceae  Oceans represent the world’s largest ecosystem, which comprise diverse visible and invisible organisms that directly or indirectly support carbon cycling in the global biosphere. The hadal trench, which is deeper than 6000 m below the sea surface, represents the most pressurized aquatic environment . Many orProchlorococcus, which is the smallest autotroph in the ocean, has been identified in the Challenger Deep of the Mariana Trench  in March 2017. Seawater samples from 4 to 4000 m were collected by Niskin Bottle mounted to a Seabird conductivity, temperature, and depth ; whereas water samples at depth of 6050 and 8320 m were collected by a self-designed acoustic-controlled full ocean depth water sampler. Five-liter of seawater from each layer was firstly filtered using 20-μm pore-size membrane filters, and then the subsamples were harvested onto 0.22-μm pore-size membrane filters . The 0.22-μm pore-size membrane filters were used in the further analysis. A total of two filters (duplicates) were prepared for each seawater sample. The filters were frozen immediately in liquid nitrogen and kept at -20°C on board. By the end of cruise, the filters were transferred to -80°C refrigerator in land-based lab until DNA extraction. The environmental parameters  were measured using a CTD sensor . Nutrients and dissolved oxygen (DO) were sampled and analyzed following the protocols described in Water samples were collected at the station Challenge Deep from 11 layers of the Mariana Trench . The procedure and analysis followed the protocol described by SRP154062.Total DNA was extracted following the method described by The raw data were filtered to eliminate low-quality reads to obtain clean reads. The raw data trimming and assembly were performed using Trimmomatic and FLASH v1.2.11, respectively . The rea1. The taxonomy classification was described according to the taxonomic description of NCBI taxonomy database. Generally, the phylum level was used as classification for eukaryotic phytoplankton. However, some phytoplankton belonged to stramenopiles (no rank), in this case, the class level were employed for taxonomy description. As for the cyanobacteria, all the identified OTUs belonged to cyanobacteria Synechococcales , the family level were employed to distinguish them. Redundancy analysis (RDA) was conducted using CANOCO . Core species were defined as the species that were present in all sampled layers, and specific species were defined as the species that were only present within a specified water zone. The circos analysis for core species was done via online tool on http://circos.ca/.For the taxonomic annotation, the representative 18S rRNA gene OTU sequences were annotated by comparison against the SILVA database with a similarity cutoff value of 0.7 . The p23Supplementary Table S1), and these included samples from the epipelagic , mesopelagic , bathypelagic , and hadal (6050 and 8320 m) zones. The temperature gradually decreased from the surface to the hadal zone, and these results were similar to those of a previous observation . The DO concentration was 197.50 μM/L at 100 m, and DO decreased to 84.92 μM/L at 1000 m, where an oxygen minimum zone exists and was firstly reported by Figure 1A). The concentration of nitrate (NO3-) was 0.09 μM/L at 4 m, and it gradually increased to a maximum concentration of 39.35 μM/L at 1500 m before it decreased to 34.00 μM/L at 8320 m . The concentration of nitrite (NO2-) was less than 0.4 μM/L throughout the water column. The distribution pattern of the PO4 concentration was similar to that of nitrate and nitrite. The phosphate (PO43-) concentration was 0.018 μM/L at 4 m, and it reached a maximum concentration of 2.70 μM/L at 1500 m before it slightly decreased to 2.28 μM/L at 8320 m . The concentration of silicate (SiO44-) ranged from 0.55–149.61 μM/L, and the maximum concentration was detected at 3000 m ; furthermore, the silicate concentration of the Epi zone was higher than that of the other zones.A total of 11 water samples were collected from depths 4–8320 m at the same location  and minimum (2.78 × 104 cells mL-1) abundances of heterotrophic bacteria were found at depths of 4 and 8320 m, respectively. The abundance of Prochlorococcus was 1.66 × 104 cells mL-1 and 6.74 × 104 cells mL-1 at the depths of 4 and 100 m, respectively . However, the Prochlorococcus from the other nine layers  and the Synechococcus and abundance of picoeukaryotes from all 11 layers were less than 1.0 × 104 cells mL-1.The cellular abundance of PN were measured based on the differential cell types, including heterotrophic bacteria, Figure 3). The diversity pattern revealed by the 18S rRNA was quite different than that revealed by the p23S rRNA . The 18S rRNA analysis showed that the duplicates of each layer were very similar, but the p23S rRNA analysis showed a slightly different pattern. In the p23S rRNA gene analysis results, both the 2000 and the 4000 m samples displayed large differences between their duplicates, which might have been caused by sequencing bias and/or sampling. As previously reported, the technical reproducibility of the amplicon sequencing-based detection technique is quite low; however, amplicon sequencing-based detection is still useful in analyzing microbial community structure . Interestingly, the community diversity and richness values of the samples from 8320 m  were not the lowest.The phytoplankton richness and diversity were evaluated using the Chao1 and Shannon indexes, respectively . The dinoflagellates were the dominant phytoplankton between the depths of 4 and 6050 m , regardless of whether the relative abundance level or OTU level was used in the calculation; however, the dominant phytoplankton changed to Chrysophyceae at the depth of 8320 m.The results of the 18S rRNA analysis showed that the compositions of the duplicates from each layer were very similar, and a total of 169 OTUs were identified as PN. These 169 OTUs belonged to eight main phytoplankton groups , including Dinoflagellata , Chlorophyta , Haptophyta , Cryptophyceae , Chrysophyceae , Bacillariophyta , Eustigmatophyceae , Pelagophyceae , and some other groups  (Jaaginema (OTU 92) and Prochlorococcus (OTU 8). The Prochlorococcus, which contributed to approximately 62.2% of the total reads, were the predominant genus in most of the layers. Haptophyta (2.0%), Synechococcaceae (2.1%), and Eustigmatophyceae (1.8%) were also identified with relative abundances higher than 1% of the total reads . In addition, Pelagophyceae (0.4%) and Chlorophyta (0.4%) showed relative abundances higher than 0.1% but less than 1%, as determined by p23S rRNA analysis. At the OTU level, 46 OTUs and 15 OTUs contributed to Haptophyta and Chlorophyta, respectively, and Haptophyta was the most diverse group.The p23S rRNA analysis revealed 130 OTUs that belonged to phytoplankton, including species of cyanobacteria and other eukaryotic phytoplankton. Different from 18S rRNA, the p23S rRNA analysis results showed that the compositions of the duplicates from some layers were quite different, such as the samples from 1500, 2000, and 4000 m. The main differences were related to the cyanobacteria Supplementary Figure S1A and Supplementary Table S2). The 18S rRNA gene analysis revealed 33 core OTUs present in all four zones. These OTUs contained species from Dinoflagellata (23 OTUs), Pelagophyceae (1 OTU), Chlorophyta (2 OTUs), Haptophyta (4 OTUs), Cryptophyceae (1 OTU), Bacillariophytina (1 OTU), and one uncultured species. Among core species, the OTU727 assigned to the dinoflagellate Heterocapsa displayed the highest relative abundance and was followed by OTU723, which was assigned to the dinoflagellate Gymnodinium . Furthermore, 31 OTUs, 6 OTUs, 15 OTUs, and 6 OTUs specifically belonged to the Epi, Mes, Bat, and hadal zones, respectively. For the OTUs that were specific to the hadal zone, the specific species were assigned to Chrysophyceae, Dinoflagellata, Bacillariophyta, and Haptophyta.To identify the core and specific phytoplankton that were present in each water zone, the comparison analysis was constructed at the OTU level (Prochlorococcus (11 OTUs), Synechococcus (3 OTUs), Chlorophyta (1 OTU), and one unranked eukaryote . In all of these core OTUs, OTU8 (Prochlorococcus) [followed by OTU92 (Jaaginema)] was the most dominant OTU in all of the four zones . For the specific OTUs, 8 OTUs, 11 OTUs, 21 OTUs, and 5 OTUs were only present in the Epi, Bat, Mes, and hadal zones, respectively. The OTUs that were specific to the hadal zone included Coccomyxa (Chlorophyta), Chrysochromulina (Haptophyta), Ochromonas (Chrysophyceae), and Synura (Synurophyceae).In the p23S rRNA gene analysis, 27 core OTUs were identified, and they were assigned to Haptophyta (7 OTUs), Pelagophyceae (1 OTU), 4 and NO3 had a positive relationship; thus, only the PO4 was selected for further analysis. According to the RDA across all the 18S rRNA PN communities, four environmental factors, including depth, PO4, salinity, and temperature, significantly contributed to the variation in the PN communities; in contrast, NO2, SiO4, and DO had almost no correlation with the distribution of PN communities (Supplementary Table S3). RDA  showed that the first axis explained 44.47%, while the first two axes explained 65.54% of the total variation in the relative abundance of the 18S rRNA communities and 76.50% of the cumulative variation in the 18S rRNA communities and environmental factors. Chrysophyceae and Bacillariophyta were positively correlated with depth, while some groups, such as Chlorophyta and Haptophyta were significant negatively correlated with depth. Haptophyta, Chlorophyta, and Pelagophyceae were negatively correlated with salinity and PO4; however, they were positively correlated with temperature.RDA was employed to assess the relationships between the PN and environmental factors. The environmental factor correlation analysis showed that POSupplementary Table S3). The results  showed that the first RDA axis explained 34.12% of the total variation in the p23S rRNA communities. The first two RDA axes explained 50.24% of the total variation in the relative abundance of the p23S rRNA communities. Chlorophyta, Pelagophyceae, and Pseudanabaenaceae were negatively correlated with depth, and Chlorophyta and Pseudanabaenaceae were positively correlated with salinity. Haptophyta and Prochloraceae were positively correlated with DO.RDA explained 63.10% of the cumulative variation in the p23S rRNA communities and environmental factor relationships; furthermore, RDA showed that DO, depth, and salinity significantly contributed to the variation in the PN communities, while the other environmental factors had almost no correlation with the distribution of the PN communities (Supplementary Table S3). The results indicated that the depth, which was followed by temperature, was the most important environmental factor in terms of structuring the vertical distribution of PN community in the Mariana Trench. The temperatures were gradually decreased with increasing depth (Supplementary Table S1). RDA  showed that the first axis explained 36.83%, while the first two axes explained 55.03% of the total variation in the relative abundance of the 18S rRNA + p23S rRNA communities and 68.2% of the cumulative variation in the 18S rRNA + p23S rRNA communities and environmental factors. Chrysophyceae, Eustigmatophyceae, Synechococcaceae, and Prochloraceae were positively correlated with depth, while Dinoflagellata, Chlorophyta, Haptophyta, and Pseudanabaenaceae were significant negatively correlated with depth. Haptophyta, Dinoflagellata, and Prochloraceae were significant positively correlated with temperature, while Chrysophyceae, Pseudanabaenaceae, Eustigmatophyceae, Cryptophyceae, and Bacillariophyta were negatively correlated with temperature. In addition, the nutrients  and salinity also significantly contributed to the structure of the PN communities.In addition, the 18S and p23S rRNA communities were combined together, and their correlations with the environmental factors were analyzed  might be the significant environment factors that structure the PN community in the Mariana Trench. Abiotic environmental factors, such as salinity, depth, temperature, light, and nutrients, can significantly influence the physiological and ecological characteristics of phytoplankton , and its abundance was considerably less than that of Prochlorococcus. The results were similar to previous findings that stated Prochlorococcus was most abundant at the surface  and decreased with depth , and they extend much deeper in the water column, with a common maximum concentration at depths of ∼150–200 m , and the intact cells of cyanobacteria, dinoflagellates, and diatoms have been found in the deep sea at depths of 2000–4000 m . Distinct patterns and features were also revealed with other factors. For example, phototrophic pigments, including chlorophyll a and phaeophytin, were analyzed in the Challenger Deep, and the results revealed that the concentrations of these pigment were highest in the Challenger Deep compared to the shallower waters, implying that the pigment in the Challenger Deep was younger, more labile and possibly more nutritious than those at the shallower reference site (58 km from the trench and approximately 6000 m in depth) , was quite different from that in the shallower layers in this study (n depth) . The bacn depth) ; furthern depth) . In addin depth) ; howevern depth) . All of Figure 7). One part of the phytoplankton cells came from the upper layer zones via fast sinking mechanisms. These cells may sink alone or within aggregates and/or zooplankton fecal pellets, and various physical processes could help these cells sink faster into the deep sea  could be separated into at least two types (deep sea . These cProchlorococcus were present throughout the entire water column. Some PN communities, which possess the heterotrophic characteristics, e.g., Eustigmatophyceae and Chrysophyceae, displayed higher relative abundance in the hadal zone. The presence of phytoplankton cells in the hadal zone may benefit from sinking mechanism and/or heterotrophic characteristics as well as resting characteristics of the cell. The present results highlighted the distinctive biosphere characteristics of the hadal trench environment and indicated that the phytoplankton could be involved in transportation of organic matters from euphotic to hadal zone, and some phytoplankton might survive with undetermined energy metabolism in the hadal zone.The present study for the first time reported the vertical distribution pattern of PN in the Mariana Trench. The dinoflagellates and the cyanobacteria RG performed the experiments. RG, YL, YX, and LW analyzed the data. CC and RX performed sampling. SM identified the phytoplankton cells. CZ and JT provided expedition oversight. CZ and JT participated in data analysis and discussion. YL and YZ designed the experiments. RG and YZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In view of the accidents such as rock mass breakage, roof fall and coal slide in coal mines, polyurethane/mesoscopic fly ash (PU/MFA) reinforcement materials were produced from polymethylene polyphenylene isocyanate (PAPI), the polyether polyol, flame retardant, and MFA using stannous octanate as a catalyst. 3-Glycidoxypropyltrimethoxysilane (GPTMS) was grafted on MFA surface, aiming to improve the mechanical properties of PU/MFA composites. The analyses of infrared spectroscopy and compression resistance reveal that the GPTMS can be successfully attached to the surface of MFA, and the optimum modification dosage of GPTMS to MFA is 2.5 wt. % (weight percent). On this basis, the effect of GPTMS on the mechanical properties of PU/MFA reinforcement materials during the curing process was systematically investigated through a compression test, a fracture toughness test, a three-point bending test, a bond property test, and a dynamic mechanics analysis. The results show that the compression property, fracture toughness, maximum flexural strength, and bond strength of PU/MFA composites increase by 21.6%, 10.1%, 8.8%, and 19.3%, respectively, compared with the values before the modification. Furthermore, the analyses of scanning electron microscope and dynamic mechanics suggest that the coupling agent GPTMS can successfully improve the mechanical properties of PU/MFA composites because it eliminates the stress concentration and exerts a positive effect on the crosslink density and hardness of PU/MFA composites. China boasts abundant coal resources, and its annual amount of coal production is increasing year by year. However, due to the complicated geological conditions and harsh working environment in the underground coal mines, and respectively, coal-rock mass tends to become loose during roadway excavation and mining. Loose coal-rock mass can easily cause accidents such as collapse of working face, coal slide of fractured rock mass, gas outburst, and water leakage, seriously affecting the safety of underground workers and the efficiency of coal mining. As a result, researches on grouting reinforcement materials for coal mines have been gradually carried out and have achieved fruitful results after decades of application and development ,2. Due t2 released from the reaction can promote diffusion of slurry into deep pores and fractures of the coal seams and enhance the effect of grouting reinforcement [Based on the above grouting reinforcement mechanism, inorganic grouting materials such as water glass, cement, and their mixture have been adopted. While these cheap, non-toxic, flame-inhibitory, and accessible inorganic grouting materials are able to achieve strong strength of the consolidated body, they can only be applied in limited fields because of their unstable curing time, small controllable range and weak toughness . Subsequorcement . Unfortuorcement , water gorcement , and MFAorcement  into theorcement . Liu et orcement  took MFAorcement  preparedorcement . Therefoorcement ,19,20. Horcement ,22.As a compound with amphiphilic functional groups, silane coupling agent can react with both inorganic and organic substances and form “chemical bridges” to alter the interfacial adhesion between them. Besides, it can also improve the compatibility between the inorganic mesoscale particles and polymer matrix and thus greatly enhance the distribution of inorganic mesoscale particles in the polymer matrix .Hence, GPTMS was grafted on MFA surface by the surface modified technique. The modified MFA was then mixed with PU matrix to obtain PU/MFA composites. The optimal modification dosage of GPTMS was investigated, based on which the mechanical properties  of the modified PU/MFA composites during the curing process were studied. Moreover, the mechanism for the coupling agent GPTMS to effectively improve the internal crosslink density and hardness of the composites without causing stress concentration was proposed.R values range from 1.0 to 1.05)In this study, the PU/MFA composites prepared are dual-component organic/inorganic mixtures whose components are listed in In order to obtain the optimum amount of the coupling agent GPTMS, the compressive strength of PU/MFA composites modified by different contents of GPTMS  was investigated through a series of optimization experiments. The MFA was modified with different dosages of GPTMS in accordance with the following steps: First, 500 g of MFA was mixed with 200 mL of ethanol aqueous solution at a volume ratio of 9:1. Next, different dosages of GPTMS were added in order and then stirred at the speed of 600 rpm for 2 h. Finally, the MFA was dried under the vacuum environment of 50 °C for 24 h, after which it was taken out and sealed for storage.3, length × width × height), the three-point bending specimen  and the bond area (12.5 ± 0.25 mm) of iron batten  were also solidified for different times  under a dry and closed environment of 23 ± 1 °C. The specimens were prepared for a fracture toughness test, a three-point bending test, a dynamic mechanics analysis, and a bond strength test, respectively.The schematic diagram of PU/MFA composites preparation is shown in −1.Infrared spectroscopy analysis: Characteristic functional groups of the specimens were detected by a Fourier Infrared Spectrometer . Infrared spectrum testing was conducted on the MFA modified by different contents of GPTMS  and the unmodified MFA. The powdered specimens and dry potassium bromide (KBr) powder were mixed at a ratio of 1:150 and compressed into tablets. Changes of peaks in infrared spectrum were observed through scanning, and meanwhile the organic functional groups were analyzed. In the test, the measurement range was set to the wavelength of 500–4000 cmScanning electron microscope (SEM) analysis: Morphologies of the specimens were observed by a high-magnification SEM . Under the acceleration voltage of 25 kV, 2.5–5 mm cubes were taken from the treated standard specimens and dried to constant weights. The fracture surfaces of the block specimens were coated with gold in a SBC-12 ion sputtering apparatus.3) and three-point bending configurations (span: 50 mm), were carried out using air as the medium. The DMA spectra, which could reflect changes in the storage modulus E and the mechanical loss factor tan δ under the action of temperature (gT), were measured in the temperature range of 30–180 °C with a heating rate of 5 °C/min at a test frequency of 1 Hz.Dynamic mechanics analysis (DMA): The dynamic mechanical properties of PU/MFA specimens were studied by using a dynamic mechanical analyzer . The DMA measurements, which adopted rectangular specimens  are presented in 950 cm−1 a. For MFcm−1 (−1 b. Moreovt of MFA ,25. Figuar chain ,27. BesiCompressive strength mainly reflects the mechanical strength of coal-rock mass reinforcement material. Therefore, the study on pressure-bearing capacity of the composites plays a decisive role in enhancing its mechanical support . First, The storage modulus can reflect the hardness of the composites to a certain extent . Figure Tg raises. This shows that the higher the temperature is, the greater the internal friction of the material gets. The peak value of tan δ corresponds to a temperature between 60 and 90 °C, which can be considered as the glass transition temperature of the hard segment of the material. At this time, the composite materials undergo glass transition, during which the molecular motion energy increases. When the barrier is conquered, the internal motion unit of the molecule is in an activated state and the movement of the segment occurs [t occurs . FurtherIn this study, a PU/MFA reinforcement material was prepared by modifying the MFA by 2.5 wt. % GPTMS and compositing it with PU. After the modification, its mechanical properties have been greatly improved. To be specific, its compressive strength, fracture toughness, maximum flexural strength and bond strength increase by 21.6%, 10.1%, 8.8%, and 19.3%, respectively. The results of SEM and DMA further verify that GPTMS promotes the change of MFA surface from hydrophilicity to lipophilicity, eliminates the interfacial difference between organic and inorganic phases, improves the crosslink density and avoids stress concentration. As a result, GPTMS successfully improves the mechanical properties of PU/MFA composites and also positively influences the hardness of the composites. The results of microscopic mechanism analysis show that an end of the GPTMS molecule was cross-linked with MFA surface by hydrolysis, dehydration, and condensation, while the epoxy functional group at the other end was cross-linked with organic matrix through ring-opening reaction. Therefore, the coupling agent GPTMS further broadens the application of PU/MFA composite materials in coal mines. Even in harsh coal mine conditions, it can be effectively applied to the reinforcement of coal-rock mass.


Diabetes is becoming one of the most widespread health burning problems in the elderly. Worldwide prevalence of diabetes among subjects over 65 years was 123 million in 2017, a number that is expected to double in 2045. Old patients with diabetes have a higher risk of common geriatric syndromes, including frailty, cognitive impairment and dementia, urinary incontinence, traumatic falls and fractures, disability, side effects of polypharmacy, which have an important impact on quality of life and may interfere with anti-diabetic treatment. Because of all these factors, clinical management of type 2 diabetes in elderly patients currently represents a real challenge for the physician. Actually, the optimal glycemic target to achieve for elderly diabetic patients is still a matter of debate. The American Diabetes Association suggests a HbA1c goal <7.5% for older adults with intact cognitive and functional status, whereas, the American Association of Clinical Endocrinologists (AACE) recommends HbA1c levels of 6.5% or lower as long as it can be achieved safely, with a less stringent target (>6.5%) for patients with concurrent serious illness and at high risk of hypoglycemia. By contrast, the American College of Physicians (ACP) suggests more conservative goals (HbA1c levels between 7 and 8%) for most older patients, and a less intense pharmacotherapy, when HbA1C levels are ≤6.5%. Management of glycemic goals and antihyperglycemic treatment has to be individualized in accordance to medical history and comorbidities, giving preference to drugs that are associated with low risk of hypoglycemia. Antihyperglycemic agents considered safe and effective for type 2 diabetic older patients include: metformin (the first-line agent), pioglitazone, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists. Insulin secretagogue agents have to be used with caution because of their significant hypoglycemic risk; if used, short-acting sulfonylureas, as gliclazide, or glinides as repaglinide, should be preferred. When using complex insulin regimen in old people with diabetes, attention should be paid for the risk of hypoglycemia. In this paper we aim to review and discuss the best glycemic targets as well as the best treatment choices for older people with type 2 diabetes based on current international guidelines. Life expectancy is defined as the average number of years that a newborn is expected to live assuming that current mortality rates remain the same throughout its life. Global average life expectancy has increased by 5.5 years between 2000 and 2016, with the fastest increase since the 1960s, as a consequence of declining number of deaths from infectious causes . The proDiabetes is recognized as an important cause of premature death and disability. In the past three decades the age-standardized prevalence of diabetes has risen substantially in countries at all income levels; 40% of this increase is estimated to result from population growth and aging . TherefoType 2 diabetes represents the most common metabolic disease in older adults. According to the latest estimates of the International Diabetes Federation (IDF), diabetes shows a high prevalence in people older than 65 years . In 2017The number of deaths caused by diabetes in the age range of 60–99 years in 2017 was 3,200,000, which represents ~60% of deaths due to diabetes among the age group between 18 and 99 , and manSeveral factors participate in the pathophysiology of diabetes in older age. Chronological age per sè represents a risk factor for many chronic diseases . AdvanceDiabetes onset in elderly usually manifest with vague and not specific symptoms, such as dehydration, dry mouth, confusion, fatigue, lethargy, weight loss, and an increased tendency toward genitourinary infections . It has There is evidence that type 2 diabetes is associated with cognitive dysfunctions. Older diabetic patients have higher risk to develop mild cognitive impairment (MCI), all-cause dementia and Alzheimer's disease . SpecifiType 2 diabetes in elderly is a powerful risk factor for functional limitations, frailty, loss of independence, and disability . MoreoveUrinary incontinence is a frequent comorbidity of diabetes, although it is usually not-reported by patients . TherefoBoth overtreatment and polypharmacy are very common among frail older diabetic subjects. The prevalence of polypharmacy regimen, defined as the use of more than 5 medications, increases with age. Results from a Dutch study revealed that 64 persons (20%) out of 319 type 2 diabetic patients aged ≥70 years were overtreated and frail . FurtherOlder patients represent a very heterogeneous and challenging population concerning diabetes care and treatment. While treating diabetes in elderly, clinicians should be always aware of maintaining a good quality of life. Patient-centered glycemic targets are needed in order to achieve the glycemic control avoiding dangerous or extreme glucose excursions. Elderly patients are highly vulnerable to hypoglycemic events, as a consequence of progressive age-related decrease in β-adrenergic receptor function. Indeed, hypoglycemia in older age has been associated with an increased risk to develop cognitive impairment, dementia, all-cause hospitalization, and all cause mortality –44. Use P = 0.04) and increased hypoglycemic events . On the other hand, a large observational study reported that an HbA1c level > 8% was associated with increased risk of all-cause, cardiovascular, and cancer mortality in older adults with diabetes  for diabetes treatment in older adults does not allow to clearly establish the most appropriate therapeutic goals in the elderly. Three major high-profile trials  –49 condudiabetes . Actualldiabetes . Howeverdiabetes  in old dDiffering from ADA, the American Association of Clinical Endocrinologists (AACE) advises an HbA1c goal of 6.5% or lower for most patients without history of cardiovascular diseases (CVD) as it can be safely achieved, whereas, a broader HbA1c target (>6.5%) is suggested for older patients with concurrent serious illness, high risk of hypoglycemia, and limited life expectancy, as the patient does not experience characteristic hyperglycemic symptoms  .On the other hand, the American College of Physicians (ACP) suggests more conservative goals (HbA1c levels between 7 and 8%) for most older patients, and a less intense pharmacotherapy when HbA1c ≤ 6.5% . MoreoveStudies comparing the effectiveness of anti-diabetes drugs in elderly are lacking, due to the exclusion of older diabetic adults from RCTs, given the high number of comorbidity and their enhanced cardiovascular risk. Every therapeutic strategy should be chosen considering age, health status, self-manageability, cognitive and nutritional status, and comorbidities . General2, while discontinuation is recommended if GFR < 30 mL/min/1.73 m2 . Pioglitazone is the only one remaining drug of this class, as it has proven to be safe in the presence of cardiovascular disease . It is cSulfonylureas are an insulin secretagogue class, which act by favoring β-cells membrane depolarization and consequently insulin secretion. They are characterized by high glucose lowering efficacy and low cost, but they should be used with extreme caution because of the high risk of hypoglycemia and weigh gain. Short acting ones with lowest hypoglycemic risk, such as gliclazide, should be preferred in older diabetic patients, when initial therapy with metformin is contraindicated or not tolerated . By contMetiglinides are short-acting insulin secretagogue agents, that enhance early phase of insulin secretion at meals, lowering postprandial glucose levels. They present lower risk of hypoglycemia than sulfonylureas, since their activity is dependent on the presence of glucose . RepagliDipeptidyl peptidase 4 (DPP-4) inhibitors belong to the class of incretin enhancer agents. They inhibit the DPP-4 enzyme, thereby extending the life-time of GLP-1 and increasing insulin secretion in a glucose dependent manner. Drugs in this class are generally well-tolerated in older people, with neutral effect on body weight and very low risk of hypoglycemia , 67. DPP2, due to safety concerns and lack of dedicated study in diabetic population with severe chronic renal disease. If SGLT-2 inhibitors are used in combination with diuretics, lowering the dose of diuretics is needed to minimize the risks of hypotension and dehydration  inhibitors are the latest marketed oral anti-hyperglycemic agents in diabetes management. These molecules act with an innovative and different mechanism of action: they inhibit Na/glucose renal cotransporter on kidney proximal convoluted tubule, increasing urinary glucose concentration, and favoring osmotic diuresis (diuretic effect). Beyond glucose lowering efficacy, SGLT-2 inhibitors have also beneficial effects in reducing body weight and blood pressure. Their use is permitted until GFR ≥ 30 mL/min/1.73 mydration . SGLT2-iydration , 81 thatydration . Moreoveydration . Based oydration . In the ydration , 84, botr causes . Conversr causes , on the r causes . The incr causes . Therefor causes , 90.Glucagon-like peptide 1 receptor agonists (GLP-1RAs) are innovative and pleiotropic drugs that act by promoting insulin secretion and reducing glucagon secretion in a glucose dependent manner and favoring weight loss. As they use the injectable way of administration, they require neuro-psychological and physical integrity. GLP-1RAs are highly effective in lowering glucose levels, with minimal risk of hypoglycemia , 92. RecFree and fixed-ratio combinations of GLP-1RAs and basal insulin formulations have been approved by regulatory agencies to potentiate antihyperglycemic effects and glycemic control in type 2 diabetic patients , 100. AtInsulin remains the most effective drug for type 2 diabetes . The maiOlder adults with type 2 diabetes represent a complex and heterogenous age group. Managing diabetes in older age remains an important clinical challenge for all physicians, either primary care providers or specialists. As older diabetic patients present frequently frailty and/or multiple comorbidities, an individualized patient-centered glycemic target is needed in order to achieve a glycemic control avoiding dangerous hypo- and hyperglycemic events. A comprehensive geriatric assessment should be performed at diagnosis of diabetes to better understand cognitive, visual and motor abilities, and coexisting comorbidities. In the choice of anti-hyperglycemic strategies, drugs with proven tolerability, safety, and minimal hypoglycemic risk should be preferred. Anti-diabetes treatment regimens in elderly must be simple, sustainable, and safe to best mirror patients' preferences, wishes, and needs.GB, MIM, KE, and DG conceived the manuscript. ML, GB, and MIM drafted the manuscript. JM, KE, and DG reviewed and edited the manuscript. All authors gave the approval to the final version of the manuscript.MIM received a consultancy fee from MSD and has held lectures for Sanofi, Astrazeneca, and Novo Nordisk. JM has held lectures for Astra Zeneca, Boehringer-Ingelheim, Eli Lilly, MSD, Novo Nordisk, Sanofi, and Servier and received research support from Boehringer-Ingelheim, MSD, Novo Nordisk, Sanofi. KE received a consultancy fee from Eli Lilly and has held lectures for Eli Lilly, Sanofi, and Novo Nordisk. DG received a consultancy fee from Eli Lilly and has held lectures for Eli Lilly and Sanofi. The remaining 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.


Radiofrequency (RF) treatment is an invasive and promising procedure in the treatment of osteoarthritis (OA). A meta-analysis based on 12 RCT studies was to investigate whether invasive RF treatment is more effective in relieving knee pain and improving knee function. Relevant studies were searched on database of Pubmed, Embase, EBSCO, Cochrane library, Wanfang digital database, VIP database, and CNKI up to January 2018. A total of 841 participants from 12 publications were included. The weighted mean difference (WMD) and the corresponding 95% CIs were used to evaluate the difference in pain scores and OKS/WOMAC scores between RF treatment and control groups. The statistical analysis was performed by Stata 12.0. The pain scores (VAS) in the RF group were lower than those in the conservative treatment group after 1 week , 1 month , and 3 months  of treatment, while there was no significant improvement in knee function. In subgroup analyses by site of radiofrequency, RF mode showed some discrepancies in the WMD of VAS between the treatment and control groups. In addition, subgroup analysis and meta-regression showed that the efficacy of RF treatment for reducing pain is reversely related to female ratio, and we did not find any surgery-related adverse reactions. RF treatment significantly reduces the knee pain, but rarely improves the knee joint function. Radiofrequency ablation has better efficacy than pulsed radiofrequency ablation in reducing pain. Furthermore, subgroup analysis and meta-regression suggested that women are more sensitive to RF treatment than men. Osteoarthritis (OA) is a common chronic noninflammatory joint disease characterized by articular cartilage damage, which involves the subchondral bone, synovium, and periarticular tissues. The probability of suffering from symptomatic knee OA in elderly patients aged >65 years is 20–30% . CurrentInvasive radiofrequency treatment can fill the gap between conservative treatment and TKA. It is a promising procedure with advantages of minimal invasion, rapid recovery, and few adverse events, including radiofrequency ablation (RFA) on the genicular nerve, intra-articular pulsed radiofrequency ablation (PRF), and cooled radiofrequency ablation (CRF). It can be applied to patients with severe complications, who cannot undergo TKA, those with severe pain around the knee joint area after TKA, those with severe joint pain who refuse to undergo surgery, and those who have failed oral NSAIDs or knee intra-articular injection.The exploration of RFA on the articular nerve was first proposed by Choi et al. . Their tThe electronic databases, Pubmed, Embase, EBSCO, and Cochrane Central of Controlled Trials , as well as Wanfang digital database, VIP database, and CNKI (Chinese database), were searched to identify eligible studies since inception to January 2018. An in-depth literature search was performed using the following keywords: “knee pain” or “chronic pain” or “radiofrequency” or “genicular nerve” or “nerve ablation” or “intra-articular” or “thermal radiofrequency” or “knee osteoarthritis”. Each search was limited to human clinical trials in English and Chinese languages. Initially, the studies were screened according to the title, abstract, and keywords to exclude the irrelevant studies. Then, the full text of the enrolled and potentially eligible studies was carefully screened to further identify the eligible studies. In addition, the reference lists of the retrieved studies were manually checked to find out relevant studies.(1) RCTs on clinical efficacy of invasive radiofrequency treatment for OA were performed; (2) patients in the experimental group received any of the following three modes of radiofrequency: CRF, RFA, and PRF; (3) a control group was involved; (4) the visual analogue scale/numerical rating scale (VAS/NRS) or Oxford Knee Score/the Western Ontario and McMaster (OKS/WOMAC) at different time points post-treatment were provided, with their mean and standard deviations (SD) or the standard error (SE) available to calculating the weighted mean differences (WMD) and the corresponding 95% confidence intervals (CIs).(1) Patients who had underwent knee arthroplasty and arthroscopic surgery will be excluded, though they might also suffer from serious knee pain; (2) when there were duplicate studies, only the newly published or largest sample size ones were included; (3) studies with unextractable or insufficient data were also excluded; (4) non-RCT studies were excluded; (5) unpublished studies such as conference abstracts were excluded because of their poor quality and insufficient data.Two experienced researchers used a predesigned form to independently extract data. Any uncertainty or discrepancy was resolved through discussion. The following parameters were included: name of the first author, year of publication, country of publication, location of study population, study design, Kellgren–Lawrence classification, mode of RF, operating site of RF, sample size of the experimental and control groups, age, gender, VAS/NRS, and OKS/WOMAC score at different time points before and after treatment. Since VAS and NRS are comparable to some extent, we converted NRS to VAS score in the subsequent data extraction for uniform statistical analysis.Risk of bias for each study was independently assessed by two researchers (HT and GXL) using the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions . Disagre I2statistics, P < 0.05 or I2> 50% indicated that the heterogeneity between studies was statistically significant [The WMDs and the corresponding 95% CIs were used to evaluate the difference in pain scores and OKS/WOMAC scores between the RF treatment and control groups at different time points . Heterogeneity between studies was evaluated using the Cochran Q test andnificant ; then thnificant . To obsenificant , 35. DueAccording to our searching strategy, 187 studies were retrieved. Finally, 12 RCTs fulfilled the inclusion criteria, including 9 studies from Asia, 2 from Europe, and 1 from the USA . Among tDavid et al. , Shen etThree studies by Choi et al. , Gulec eIn Choi et al.  and DaviChoi et al. , David eWe identified no other potential sources of bias.I2=94.5%, P<0.01; 1 month, I2=87.9%, P<0.01; 3 months, I2=90.7%, P<0.01), the random-effects model was used to calculate WMDs and the corresponding 95% CIs. The results showed that the pain scores of the RF treatment group were lower than those of the control group at 1 week, 1 month, and 3 months after treatment [1 week, WMD -1.77, 95% CI -2.93 to -0.61, P<0.01 , while the intra-articular PRF was slow-acting , which could relieve the pain only at 1 month , but showed an insignificant improvement at 3 months .rd month . Subgroup analysis by sex ratio showed a significant difference in pain scores between the treatment and control groups. At 1 week and 3 months, the WMD of the pain score was merely statistically significant when the sex ratio was ≥ 2 . Conversely, WMD was not statistically significant when the sex ratio was < 2 . In addition, the application of blindness and the presence or absence of concomitant therapy exerted a specific influence on the results.The RF mode was also one of the reasons for the improvement in pain. CRF (no data in 1 week) and RFA treatment showed a significant improvement in pain at all observation time points. However, PRF treatment did not show any statistically significant improvement in the pain till the 3Due to the significant heterogeneity among the study groups and limited findings from subgroup analyses, meta-regression analyses were conducted to further verify the source of between-study heterogeneity. At 1 and 3 months after treatment, the sex ratio was inversely correlated to the WMD of VAS. The slope of the meta-regression curve at 1 month was -0.67, 95% CI -1.08 to-0.27, P<0.01, which might explain 71.56% heterogeneity across studies, while that at 3 months was -0.73, 95% CI -1.48 to 0.03, P=0.06, which might account for about 36.88% heterogeneity across studies Figures .Sensitivity analysis was used to evaluate the stability robust of the pooled WMDs. After the sequential exclusion of individual studies one by one, the WMDs were recalculated to identify the significant change in our results. Sensitivity analysis the elimination of any single study was unlikely to overturn our findings -c.P=0.45; 1 month, P=0.28; 3 months, P=0.75) or Egger's regression test  did not show any publication bias in this meta-analysis.Evaluation using Begg's correlation tests  or RFA in the genicular nerves significantly improved the pain within 1 week, and the analgesic effect could maintain up to 3 months. Nevertheless, intra-articular PRF was slow-acting with a short duration, its pain relief effects appeared until 1 month, but vanished at 3 months. Thus, it is suggested that RF thermocoagulation of genicular nerves can improve the refractory pain of the knee joint and the quality of life of patients. In addition, the pain relief effects of RF treatment were inversely associated with sex ratio. The higher the female ratio was, the more effective the pain relief effects were. We did not find any surgery-related adverse reactions. Furthermore, no publication bias was found in the current study, and the sensitivity analysis suggested that the pooled effect sizes did not alter significantly after the exclusion of individual studies sequentially, which confirmed the stability of the results.β fiber). During RFA, the high temperature of the electrode tip of the RFA disrupts the nerve tissue with respect to protein denaturation and coagulation necrosis, which can significantly reduce the rest pain but have a limited effect on the activity pain [th month, which was similar to the results of the subgroup analysis in the current study. However, most of the studies mentioned above were retrospective analyses and lacked a control group, which did not allow the comparison of the curative effect with the traditional treatment. Moreover, only one RCT was related to CRF in the genicular nerves. Thus, additional clinical trials are essential for exploring the efficiency of analgesia.The mechanism of intra-articular PRF or RFA of genicular nerves has not yet been clarified; however, all published studies suggest that RF treatment can significantly reduce pain around the knee , 36–40. ity pain . The effity pain . Compareity pain , the proity pain , 28 repoIn this meta-analysis, among the 7 studies on RCT of RFA in the genicular nerves, only 2 studies had performed prognostic nerve blocks with local anesthetic, in which patients with a remission rate of > 50% were enrolled in the RFA group, and 12–25.9% showed ineffective treatment or did not respond to the treatment . 1-2 mL Based on the published studies, the three RF methods can improve the knee function , 26, 37.No surgical-related adverse reactions were reported during RF treatment group. Some studies stated severe pain during surgery, which might be attributed to the electrode tip of the needle that was close to the periosteum or tendon and could be reduced by replacing the needle site . Some stSubgroup analysis showed the sex ratio indicators of better pain improvement. The meta-regression analysis further proved that the higher the female proportion, the better the treatment effect, although the underlying mechanism is yet to be elucidated. Women are prone to suffering from chronic pain syndrome, and the frequency, areas of the body, duration, and severity of pain are significantly higher than those in men –46. In aFor all the RCTs included in our studies published in the past 9 years, their limitations are summarized as follows. (1) Some of the researches were conducted based on relatively small sample size (35 to 42 patients) , 14, 16.In the present study, we conduct a pooled analysis of improvement in the pain and knee joint function with RF treatment at different time point using meta-analysis method. The absence of published bias in the enrolled studies and sensitivity analysis indicated the robustness of the results. Nevertheless, the present study has certain limitations. Firstly, the WMDs of all combined trials showed a statistically significant reduction in VAS scores at some time points, but this difference was below the widely accepted, minimally clinically significant decrease in VAS of at least 2/10. Second, the blind method was adopted in only 3 studies, indicating a potential high risk of bias. This might be because blindness was not applicable in some studies. Third, Few or no data was related to cartilage mechanism or change in morphology. In addition, only one RCT of CRF was included in our analysis. The comparability of this treatment with other types of RF treatment needs further investigation.In summary, our meta-analysis showed that, in comparison with the conservative treatment, invasive RF treatment significantly improved the knee joint pain, but exerted a limited effect in improving the knee joint function. In addition, CRF or RFA on genicular nerves exhibited a more significant and long-lasting analgesic effect as compared to intra-articular PRF. Furthermore, women are more likely to benefit from RFA treatment on the genicular nerves around the knee joint than men. Both subgroup analysis and meta-regression suggested a positive correlation between knee pain remission and female in the gender ratio, although the mechanism is not yet clarified. Due to the limitations of included literatures, the authenticity of the results requires further verification by prospective multicenter randomized controlled trials.


Contamination of hair dyes to heavy metals can threaten consumer’s health. We investigated the concentrations of some important heavy metals in hair dyes and evaluates their non-carcinogenic effects.The most commonly used hair dyes were determined through questioners and 32 samples were collected from the market of Tehran in 2014. The concentration of 10 heavy metals  was determined using ICP-MS. Based on the obtained data from distributed questionnaires and Monte Carlo simulation, the exposure to the evaluated heavy metals was estimated. Besides, using hazard quotient (HQ) and chronic hazard (HI), the risk of non-carcinogenic effects of investigated hair dyes consumption was specified.−1 and those of Cd, Cu, and Pb as 0.45, 61.32, and 185.34 μg kg−1, respectively. Pb with HQ of 7.46e-4 had the highest risk and Fe with HQ of 3.4e–6 had the lowest level of risk. Among the investigated dyes, the ones made by Iran (HI=2.8e–4) and the dark brown color (HI=1.93e–4) had the highest level of risk among all the studied samples.Results indicated the average concentrations of Al, Ba, and Fe as 0.54, 0.86, and 1.19 mg kgTwo indices of HI and HQ showed that heavy metal contents in the investigated samples had not probable non-carcinogenic risks for the consumers of these products. Heavy metals are one of the most important environmental pollutants, some of which even at very low values could be dangerous for human health because they have long biological half-lives, are non-biodegradable, and some are toxic even at very low concentrations , 2. PeopOn the other hand, cosmetics products since the dawn of civilization are considered a part of routine body care . Direct Among different cosmetic products, hair dye is one of the most common products in the market. Skin is the direct way for entering the components of hair dyes . Hence, Hence, in the current study, we determined the concentration of some heavy metals in the most widely used brands of chemical hair dyes in the market of Tehran, and then, we estimated the exposure levels of female residents of Tehran to these elements; and subsequently, the related non-carcinogenic health risk to the consumption of these products was evaluated.To determine the concentration of some important heavy metals in chemical hair dyes, in 2014, 8 widely used brands were selected from market of Tehran and, in each brand, 4 types of colors  were selected. The sampled brands were made by Iran, Spain, Italy, and Germany, although some of these brands probably were fake brands. The brands were gathered from different stores in the market of Tehran City randomly.3 (nitric acid) was added to 1 gr of each sample. After heating for 4 h at 85 °C the sample was cooled down at room temperature and 1 ml of peroxide hydrogen 30% was added and it was injected to couple plasma-mass inductively spectrometry (ICP-MS) in order to determine the concentration of Fe, Ag, Co, Cr, Mn, Ba, Cd, Cu, Pb, and Al  is average daily intake of heavy metals through skin exposure to hair dyes, C (mgkg−1) is metal concentration in hair dye, SA (cm2) is the skin area of scalp, SL (mg cm−2) is skin absorption factor, ABC (unitless) is the depth absorption skin factor, EF (day year−1) is exposure frequency, ED (year) is exposure duration, BW (kg) is body weight, and AT (day) is average time. AT parameter is obtained using −1day−1) is the reference dose and HQ (unitless) is hazard quotient. Moreover, HI (chronic hazard index) is obtained by In order to evaluate exposure levels to heavy metals due to hair dyes consumption, 370 questionnaires were distributed in female citizens of Tehran and some key parameters such as weight, age of starting the use of chemical hair dyes, and frequency of hair dye consumption was questioned. Besides, Risk assessment process is related to uncertainty that might arise from uncertainty in the measurement or estimation of parameters . Hence, Findings from the distributed questionnaire showed that 92.19% of the participants used hair dye. In addition, about 19.5% of female citizens in Tehran started using this product from the age of 12 yr old, while most of the participants (34.7%) started using this product from the age of 19 yr old. Moreover, about half of the participants (49.5%) declared that they use chemical hair dyes. Furthermore, 4 colors  had the highest consumption rate as 33.6%, 27.4%, 19.6%, and 14%, respectively. Moreover, based on the questionnaire, 8 brands were determined as the most widely used hair dyes; the countries which made those brands were as follows: Italy (19.7%), Iran (13.6%), Spain (8.3%), Iran (6.8%), Spain (6.8%), Iran (5.3%), Italy (5.3%), and Germany (5.3%). Four colors of each most widely used hair dye brands were purchased and were transferred to the laboratory for heavy metal measurement.According to The average daily intake of heavy metals through skin exposure to investigated chemical hair dyes was evaluated using Monte Carlo simulation, for this propose, 20000 iterations were utilized to calculate dermal in order to determined risk characterization  is shown in dermal values of evaluated elements among different brands and different colors were presented in Central index of median in all the simulations had the highest probability than other central indices, hence, we used median of the Drization . Besidesdermal  on the DIran is the second biggest consumption market for cosmetic products in the Middle East . Chemica−1 in the investigated brands and colors; besides, the average of it was found 0.54±0.64 mg kg−1. Compared with other studies, the rate of this metal in all the collected samples was too low; for example, in Saudi Arabia on mascara and eye shades, the concentration of Al was reported as 50 mg kg−1 , the average of this metal was reported as 0.000238±0.000001 mg kg−1 which it was lower than our findings  (Exposure to Cd could affect the cardiovascular system and vessels . Moreovefindings . In anotfindings . The con (Henna) .Exposure to Cu could threaten the liver and create Wilson’s disease and insomnia . In addiAverage concentration of this metal in the analyzed samples was 61.32±100.59 ppb. This value was less than other findings , 14; the−1, and it was ranged from 0.17 to 11.58 mg kg−1 in different colors and brands. In compare to our findings, the average of Fe determined in hair pomade as 209.8 mg kg−1  have been completely observed by the authors.


To investigate the effects of maternal preeclampsia on inflammatory cytokines and neonatal outcomes in premature infants.The study included preterm infants born at gestational age ≤32 weeks in a tertiary university hospital between January 2016 and January 2017. The study group consisted of infants born from mothers with preeclampsia (Group-1), and the control group consisted of infants born from normotensive mothers (Group-2). Demographic characteristics and clinical outcomes of the infants were recorded. IL-6, IL-8, IL-10, and TNF-α cytokine levels were measured from umbilical cord blood samples.A total of 108 infants were included in the study, of which 34 were in the Group-1 and 74 in the Group-2. Gestational ages (29 vs 30 weeks) of the infants in both groups were similar. There was no significant difference between the cytokine levels of infants with and without preeclampsia. The rate of small for gestational age, retinopathy of prematurity, intraventricular hemorrhage, necrotizing enterocolitis, neutropenia, and thrombocytopenia were significantly higher at the infants with preeclampsia.Maternal preeclampsia leads to an increase at the neonatal morbidities in premature infants without causing a significant alteration at the cytokine levels in cord blood. Preeclampsia, which may lead to maternal and perinatal morbidity and mortality, affects 1-7% of pregnancies.2Cytokines are the general name of small protein structure molecules involved in immune, inflammation, and hematopoiesis and are divided into two as proinflammatory and anti-inflammatory effects.3Different results have been reported on inflammatory cytokines which play an essential role in preeclampsia.3We have hypothesized that preeclampsia may alter the balance between anti-inflammatory and proinflammatory cytokine levels in premature babies. In this prospective study, we aimed to investigate the effects of maternal preeclampsia on inflammatory cytokines and neonatal outcomes in premature infants.This study was conducted at a tertiary university hospital between January 2016 and January 2017. Premature infants born at a gestational age of ≤ 32 weeks were included in the study. Written and verbal consent was obtained from the parents. Infants who were born to mothers with diabetes mellitus, chorioamnionitis, and early membrane rupture were excluded from the study. Babies with early sepsis (who had high acute phase reactants in the first 72 hours or positive blood culture) and who had chromosomal or major congenital anomalies were excluded from the study. A flowchart with the participation of patients in the study is shown in The study was approved by the ethics committee  of Uludag University Medical Faculty.Clinical features including gender, birth weight, gestational age, type of delivery, neonatal morbidities, antenatal steroid application and preeclampsia were recorded. The study group consisted of babies with preeclampsia and the control group consisted of babies with normotensive mothers. After 20 weeks of gestation, ≥140 / 90 mm Hg blood pressure with 300 mg/day proteinuria was defined as preeclampsia.2Respiratory distress syndrome (RDS)Gestational ages were evaluated according to modified Ballard scoring.12Umbilical cord blood samples were taken immediately after delivery. These samples were kept for 30 minutes and then centrifuged at 2000 x g for ten minutes. Serum samples were stored at -20 °C until measurement. IL-6, IL-8, IL-10, and TNF-α cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA).3.The leukopenia and thrombocytopenia were evaluated by leukocyte and platelet counts on the 1st-day complete blood count of the patients. Thrombocytopenia was defined as a platelet count of <150.000/mm.The findings of the study were analyzed using the SPSS version 23 program with an appropriate statistical method. Chi-square test was used for categorical variables. Shapiro-Wilk test was used to compare the distribution of two independent samples in groups, while those with normal distribution were compared with the t-test and those without a normal distribution were compared with the Mann Whitney U test. P values <0.05 were considered to be statistically significant.During the study period, 108 infants who met the study criteria of 204 premature infants born at ≤32 weeks of gestational age were included in the study . DemograIn the study and control groups, umbilical cord blood cytokine levels and leukocyte, neutrophil and platelet counts on day one are given in Preeclampsia continues to be one of the important causes of perinatal mortality and morbidity.Different results have been reported in studies that investigate the clinical effects of maternal preeclampsia on premature infants. In the literature, there are different studies reporting that birth weight and Apgar scores are lower in babies of preeclamptic mothers while thrombocytopenia, neutropenia, SGA, ROP, BPD and NEK ratios are higher.Preeclampsia can have different effects on more than one system.20Also, there is a positive correlation between IL-6, IL-8, and TNF-α.IL-10 plays a crucial role in the physiopathology of preeclampsia as an immunomodulator. Low levels of IL-10 were associated with preeclampsia.16It includes a limited number of cases. Another limitation of this study is that we measured a small number of cytokine levels as a marker of inflammation.As a result of maternal preeclampsia, birth weight and APGAR scores of preterm infants were significantly lower, and length of hospital stay, SGA and ROP rates were higher. Besides, the rates of IVH (41% vs. 27%) and NEC (18% vs. 8%) were higher in the infants of preeclamptic mothers, although not statistically significant. However, levels of TNF alpha, IL-6, IL-8 and IL-10 in premature infants were found to be unaffected by the mother’s preeclampsia condition. In this study, it was shown that maternal preeclampsia leads to an increase at the neonatal morbidities in premature infants without causing a significant alteration at the cytokine levels in cord blood. Studies are needed to explain the physiopathological basis of the adverse clinical effects of preeclampsia in premature infants.SCC, BAD, HO, NK: Conceived, designed and did statistical analysis & editing of manuscript, are responsible for integrity of research.SCC, BAD: Did data collection and manuscript writing.BAD, SCC, HO, NK: Did review and final approval of manuscript.


Co-infection between malaria and HIV has major public health implications. The aims of this study were to assess the malaria prevalence and to identify predictors of positivity to malaria Test in HIV positive patients admitted to the health center São Lucas of Beira, Mozambique. A retrospective cross-sectional study was performed from January 2016 to December 2016. Overall, 701 adult HIV patients were enrolled, positivity to malaria test was found in 232 (33.0%). These patients were found to be more frequently unemployed (76.3%), aged under 40 (72.0%), with a HIV positive partner (22.4%) and with a CD4 cell count <200 (59.9%). The following variables were predictors of malaria: age under 40 , being unemployed , irregularity of cotrimoxazole prophylaxis’s , CD4 cell count <200  and tuberculosis comorbidity . In conclusion, high malaria prevalence was found in HIV patients accessing the out-patients centre of São Lucas of Beira. Our findings allowed us to identify the profile of HIV patients needing more medical attention: young adults, unemployed, with a low CD4 cell count and irregularly accessing to ART and cotrimoxazole prophylaxis. Plasmodium spp. and, consequently, leading to a more frequent occurrence of clinically severe malaria cases [Despite improvements, malaria and acquired immune deficiency syndrome (AIDS) are two of the most important infectious diseases worldwide . Malariaia cases . Interesia cases .Plasmodium falciparum infection accounts for 90% of all malaria cases, followed by P. malariae and P. ovale responsible of about 9% and 1%, respectively. In 2015, the confirmed cases of malaria were 8,520,376 [Mozambique is one of the sub-Saharan African countries with the highest incidences of HIV co-infection associated to endemic malaria ,6. In MoHowever, the number of studies on prevalence and clinical manifestations of HIV- malaria co-infection in Mozambique is limited ,8. The aA retrospective observational cross-sectional study was designed and implemented to analyze data of patients accessed the health centre of São Lucas of Beira, Sofala, Mozambique, from January 2016 to December 2016.The health center of São Lucas provides access to free care and treatment of HIV/AIDS patients in an out-patients setting. For each new admitted patient, medical history is collected and the HIV status is checked with the HIV Rapid Test and, if positive, confirmed by western blot. Each consecutive consultation includes full clinical examination, (including HIV status according to WHO), ART therapy, cotrimoxazole prophylaxis, CD4+ T cell count, partner HIV status, co-morbidities  and other sexual transmitted infections (STI), including genital herpes, condyloma, syphilis, gonorrhea and candidiasis. With regard to ART therapy and cotrimoxazole prophylaxis, information on regularity of their administration are also collected. In case of clinical suspicion of diabetes or tuberculosis, specific diagnostic protocol according to WHO guidelines is applied to confirm the diagnosis .Malaria screening is performed using the Malaria Rapid Diagnostic Test (RDT) kit, as described by the manufacturer’s . The confirmation of RDT results is obtained by blood smear microscopy . All patA sample size estimation was performed using the following formula :(1)n=Z2x): 25.9% , e = errA convenience sampling of at least 298 PLWHIV was estimated. All HIV/AIDS adult patients (>18 years) that consecutively accessed the health center for a medical consultation or to collect their antiretroviral (ARV) drugs from January 2016 to December 2016 were identified through the patients’ registry and were recruited in the study.p-value < 0.10 at the univariate analyses were included. Multicollinearity among covariates was assessed through the variance inflaction factor (VIF), taking a value of 2 for excluding a covariate. However, no variable was excluded according to the previous criterion. Odds Ratios (ORs) as adjusted Odds Ratios (Adj-ORs) with their 95% confidence intervals (CIs) were used to measure the association between factors at the baseline (exposure) and Malaria positivity (outcome).Categorical variables were reported as absolute and relative frequencies (percentages). Chi-square test (with the Yates’ correction as required) was used to compare categorical variables. A logistic regression model was implemented as follows: Malaria positivity was considered as dependent variable and each one of the available factors at the baseline evaluation were used as independent variables . In the multivariate analysis all the factors with a p-value < 0.05. Analyses were performed by using the SPSS 21.0 for Windows .All statistical tests were two-tailed and statistical significance was assumed for a Ethical approval of the protocol was achieved by District Health Authority in Beira, the Health District Direction (protocol reference: 189/17), Mozambique and (as this study used secondary data) informed patient consent was not required.n = 430, 61.3% females; n = 421, 60.0% under 40 years old) were enrolled in the study. The demographic and HIV related characteristics, overall and according to the malaria test result (positive versus negative), are summarized in n = 177; 76.3%), aged under 40 , with a HIV positive partner  and with a CD4 cell count <200 , being all of these differences statistically significant . By contrast, malaria negative patients resulted were more frequently receiving cotrimoxazole prophylaxis , under ART , and to be more regular in taking it ART  . No statistical differences between the two groups were observed for gender  and HIV stage defined by the WHO .A total of 701 adult HIV positive patients . Conversely, no statistically significant difference was highlighted between the two groups for hypertension, diabetes and other STIs.The multivariate model considered the effects on malaria (dependent variable) of age (<40 years old), occupational status (unemployed), partner HIV positivity, being under ART, regularity of ART, CD4+ T cell count < 200, being under cotrimoxazole prophylaxis, irregularity of cotrimoxazole prophylaxis and presence of tuberculosis comorbidity. The following variables resulted predictive of malaria positivity : age undThis cross-sectional study aimed to estimate the prevalence of malaria and to identify its predictors in HIV patients accessing to the health center of São Lucas of Beira, an endemic malaria area. Malaria and HIV are two of the most challenging global health issues for developing countries, especially for Mozambique, where malaria accounts for 29% of all deaths, closely followed by AIDS, responsible of 27% of the mortality in the general population ,14. MoreHowever, to the best of our knowledge, only a few studies have evaluated the epidemiological aspects of HIV and Malaria co-infection in Mozambique ,15,16,17Malaria prevalence in our study population was 33.0%, being higher when compared to the 25.9% reported among HIV patients hospitalized at Central Hospital of Beira and to the 9.8% of HIV patients hospitalized at Central Hospital of Maputo in 2008 . In the Moreover, although it is well known than HIV patients are more susceptible to malaria, in this study we identified a further more vulnerable group, consisting of young and unemployed patients with a low CD4 cell count, irregular in taking cotrimoxazole prophylaxis and with a previous history of tuberculosis. This evidence is in line with the current literature ,21. In fAgain, according to the literature, the patients enrolled in our study undergoing regularly to ART were less susceptible to malaria. This effect could be explained by the non-adherence to ART that can increase the risk of opportunistic infection such as malaria, being included in the list of AIDS-related opportunistic infections by CDC since 2009 ,26. LikeAnother aspect emerging from our data is the role played by tuberculosis and health determinants such as socio-economic status in the interaction between HIV and malaria . In factThe major limitations of this study are related to the cross-sectional design and to the typology of patients included in the study: since the study enrolled non-hospitalized patients, the possibility of self-medication for malaria could have biased the prevalence detected. In addition, the lack of data on pregnancy status, haemoglobin values and other health outcomes, did not allow a more comprehensive interpretation of results, so when HIV and malaria infection occur together a higher risk of complications should be considered.Our findings allowed us to identify the profile of HIV patient needing more medical attention: young adults, unemployed, with a low CD4 cell count and irregularly accessing to ART and cotrimoxazole prophylaxis.Despite the noted limitations, our study documented a very high malaria prevalence in a population of HIV subjects accessing an out-patients center in Mozambique, allowing us, at the same time, to better understand the profile of these patients that are highly vulnerable to co-infection. These findings could assist decision makers in efforts to plan HIV and malaria prevention interventions in low income countries and in settings with high endemicity. In particular, a more extensive use of cotrimoxazole for preventing and protecting opportunistic infections associated to malaria should be advocated with regard to young and unemployed HIV patients.


Background: Dunnigan-type familial partial lipodystrophy (FPLD2) is a rare autosomal dominant disease caused by heterozygous mutations in the LMNA gene that results in regional loss of subcutaneous adipose tissue with onset in puberty. However, a generalized lipodystrophy phenotype has also been associated with heterozygous mutations in this gene, demonstrating the noticeable phenotypic heterogeneity of this disease.Methods: We report and describe clinical and metabolic features of four patients from the same family with the p.R582C LMNA mutation, three homozygous and one in the heterozygous state that present with three distinct lipodystrophic phenotypes.Results: Case description: The proband was a 12-year-old girl who developed severe subcutaneous fat atrophy in limbs and abdomen followed by a remarkable dorsocervical fat accumulation in adulthood along with diabetes at age 23. The proband's sister was a phenotypically normal girl who developed hypertriglyceridemia at age 8, progressive features of partial lipodystrophy at age 11, and diabetes at age 22. The proband's mother was first examined at age 32, presenting diabetes and a severe generalized lipodystrophic phenotype; she developed kidney failure at age 41 and died due to diabetic complications. The proband's father was a 50-year-old man with abdominal fat concentration that was initially considered phenotypically normal. Massively parallel sequencing using a platform of genes related to genetic lipodystrophies, followed by Sanger sequencing, revealed the transversion c.1744C>T at exon 11 of the LMNA gene (p.R582C) in the homozygous (mother and daughters) and heterozygous (father) states.Conclusion: We documented three distinct phenotypes of the homozygous and heterozygous p. R582C LMNA mutation in the same kindred, illustrating that FPLD2 linked to mutations in this gene is a disease of great clinical heterogeneity, possibly due to associated environmental or genetic factors. Dunnigan-type familial partial lipodystrophy  is a rare autosomal dominant disease characterized by regional loss of subcutaneous adipose tissue (limbs and trunk) that occurs progressively during peripubertal phase and is often associated with conspicuous insulin-resistance, diabetes, and other metabolic disturbances later in life (LMNA gene (MIM150330) on chromosome 1q21–22  or impair the capacity of lamins in interacting with transcriptional regulators (gene expression model)  . FPLD2-cn model) . The hetin FPLD2 , 15.LMNA mutation in the homozygous state  and a less severe phenotype in a heterozygous carrier in the same family.In this report, we describe two severe and distinct lipodystrophic phenotypes associated with the p.R582C Clinical features as well as the laboratory and molecular data of four related patients are presented herein. This study was approved by the ethics committee of the Universidade Federal do Ceará, Fortaleza, Brazil. The patients gave written informed consent for the study and for publication of their clinical, biochemical, and molecular data.Blood samples were obtained after overnight fast. Blood glucose, cholesterol, and triglyceride levels were determined according to standard methods using automated equipment, serum insulin levels were determined by immunoassays with reagents provided by Roche Diagnostics , and hemoglobin A1c values were measured by ion exchange high performance liquid chromatography (HPLC). Plasma leptin levels were determined by an immunoassay using a commercial kit .Body fat distribution was assessed using whole body magnetic resonance imaging (MRI) performed in a 1.5-Tesla imaging device . Pre-selected body segments were surveyed using contiguous axial and coronal 3.5-mm slices, a T1-weighted spin-echo sequence, and in-phase and out-of-phase T1 sequences for abdominal studies.AGPAT2, BSCL2, CAV1, PTRF, LMNA, PLIN1, ZMPSTE24, AKT2, CIDEC, TBC1D4, and PPARG. The panel included 51 genes related to monogenic forms of diabetes in addition to the mitochondrial genome  and thus the proband was started on levothyroxine resulting in catch-up growth and normal pubertal development with menarche by age 14.8. After hypothyroidism treatment, the selective loss of subcutaneous fat tissue in limbs, gluteal region, and abdomen became evident over time, leading to the diagnosis of lipodystrophy. At age 12, she presented with moderate hepatomegaly, umbilical hernia, hypertriglyceridemia (509 mg/dL), and acanthosis nigricans in the neck and axillary regions. One year later, at age 13, hypochromic and atrophic cutaneous plaques were observed distributed throughout the body. Skin biopsies revealed localized scleroderma (morphea). Some years later in adulthood, remarkable fat accumulation in the neck, face, and axillary and dorsocervical regions was observed, along with the worsening of subcutaneous fat atrophy in limbs and abdomen, indicating partial lipodystrophy diagnosis.The proband Figure  was a 122. A formal assessment of intelligence quotient is not available, but she shows a slight degree of intellectual impairment. Standard serum determinations are listed in Table At age 23, the proband was diagnosed with diabetes and albuminuria. Currently, she is 26 years old and presents with uncontrolled diabetes, hepatomegaly , and irregular menses. She is 145 cm in height, weighs 42 kg, and has a BMI of 20.0 kg/mThe proband's sister Figure  was born2. Standard serum determinations are listed in Table Currently, she is 22 years old and has irregular menses, hepatomegaly , and was recently diagnosed with diabetes. She is 163 cm in height, weighs 55 kg, and has a BMI of 20.7 kg/m2. She had no signs of acanthosis nigricans and her liver was palpable 3 cm below the costal margin. Serum determinations at first visit are listed in Table When the proband was first examined at our unit, a clinical examination of her mother Figure  at age 32 .Massively parallel sequencing using the platform of genes related to genetic lipodystrophies revealed that the proband's mother had a homozygous genotype for the transversion c.1744C>T located at exon 11 of the Molecular studies of the daughters' genomes were performed using Sanger sequencing, which confirmed the p.R582C variant in the homozygous state. The proband's father was also tested and was found to have the same p.R582C but in a heterozygous state.The c.1744C>T/p.R582C variant has been described as pathogenic by Mory et al.  and is rAn MRI scan was available for patient 2 (proband's sister). It showed near total absence of subcutaneous fat throughout the body in the analyzed body segments and preserved fat in the palms, soles, labia majora, and scalp. Retro-orbital fat was also present. Intraperitoneal, retroperitoneal, and perirenal fat was well preserved and there was a marked accumulation of fat in deep cervical regions as well as in the infraclavicular, axillary, and scapular regions. Bone marrow fat was markedly reduced in the spine and humerus, relatively preserved in the proximal femur, and normally preserved in the distal femur, tibia, fibula, and feet Figure .The MRI scan of patient 4 (proband's father) showed a less severe loss of subcutaneous fat throughout the body and preserved fat in the palms, soles, scalp, and retro-orbital regions. Intraperitoneal, retroperitoneal, and perirenal fat was well preserved and there was a less severe fat accumulation in the deep cervical regions in comparison with his daughter. Bone marrow fat was also markedly reduced in the spine and humerus Figure .Familial partial lipodystrophy of the Dunnigan type is an autosomal dominant disease with noticeable phenotypic heterogeneity.2, 34.6% total body fat, 32.9 μg/mL leptin, who was diagnosed with dyslipidemia and hypertension at age 36 and diabetes at age 43  of patient 3 (proband's mother) may have prevented the typical fat accumulation of FPLD2, thereby masking the disease, highlighting the diagnostic difficulties regarding nutritional status especially in underdeveloped regions where undernutrition is a problem.We identified three distinct phenotypic variants in our patients. Patient 1 and 2 (daughters) show typical partial lipodystrophy while patient 3 (proband's mother) showed generalized lipoatrophy. The generalized and partial lipodystrophic phenotypes associated with the same mutation in a homozygous state is remarkable, suggesting that other genetic factors are modulating clinical expressivity of this disease. On the other hand, it is also possible that the apparent generalized phenotype is a result of poor nutritional status as the patients come from a poor region and have a low family income. The low BMI  was a heterozygous carrier initially considered phenotypically normal. After discovering that the father harbors the same mutation in a heterozygous state, a more careful clinical evaluation revealed a less severe phenotype of partial lipodystrophy that was confirmed by MRI. This phenotype is comparable to the patient described by Mory et al. , a carriLMNA gene associated with a lipodystrophic phenotype were found in the heterozygous state. However, a few patients were described with homozygous mutations. Le Dour et al.  and HDL-cholesterol levels. This patient was classified by the authors as having typical FPLD2 and her parents were first-degree cousins heterozygous for this mutation. The father could not be evaluated, but he apparently did not exhibit the phenotypic alterations of lipodystrophy and had mild dyslipidemia . The mother had vitiligo lesions and dyslipidemia  but no evidence of lipoatrophy or acanthosis. The same p.R584H LMNA mutation has been previously described in the heterozygous state in two patients with familial partial lipodystrophy and metabolic complications such as diabetes, dyslipidemia, and hepatic steatosis , and severe metabolic disturbances; molecular studies revealed a heterozygous p.T10I mutation in the LMNA gene. Similarly, a 27-year-old man was described by Caux et al. , heterozygous mutations in the malities , 27. Mormalities  describen et al.  describeIt is worth noting that the proband's mother (patient 3) and daughters (patients 1 and 2) were homozygous for this genetic variant. The parents of patient 3 were unrelated, but they are from a small country town in the state of Ceará in northeast Brazil that has 26,000 inhabitants and where consanguineous unions are frequent. On the other hand, patient 3 had a consanguineous marriage with a second-degree cousin, explaining the two homozygous daughters (patients 1 and 2).LMNA gene mutations while WS is most frequently caused by mutations in the RECQL2 gene, which is involved in DNA repair and telomerase maintenance process. However, some atypical cases of WS have been associated with heterozygous LMNA point mutations  and Hutchinson-Gilford progeria syndrome (HGPS). HGPS has been attributed to utations . This asLMNA gene affecting the C-terminal domain and specific for the lamin A isoform. Laminopathy phenotypes may be linked to the position of the LMNA mutation in relation to the nuclear localization sequence (NLS), which spans residues 416–423. Mutations associated with familial partial lipodystrophy are mostly downstream this point and in the C-terminal domain of lamin A/C (In this study, we identified a p.R582C mutation located in exon 11 of the amin A/C ; this doamin A/C , 32.LMNA mutation in homozygous and heterozygous states in the same kindred. As this variant affects only lamin A and spares the other splice form, it may render a more severe phenotype when occurring in the homozygous state, suggesting a dose-dependent effect of the p.R582C LMNA mutation in affected patients. However, this mutation in a heterozygous state resulted in an uncertain phenotype and its homozygous state caused severe lipodystrophic syndromes with phenotypic differences. Our findings, as well as the previously described cases of generalized lipodystrophies due to LMNA mutations, illustrate that familial lipodystrophy linked to mutations in the LMNA gene is a disease of great clinical heterogeneity that is possibly due to associated environmental or genetic factors.In conclusion, we documented three distinct phenotypes of the p.R582C The protocol was approved by Walter Cantídio University Hospital Research Ethics Board, from Universidade Federal do Ceará, Fortaleza - Brazil. This study was carried out in accordance with the principles of the Declaration of Helsinki and written informed consent was obtained from all subjects.RM and Cd contributed to the conception of the work, acquisition, and interpretation of data, as well as writing and the manuscript review. AJ and MT contributed to the analysis and interpretation of data and reviewed the manuscript. VF, AM, and LK contributed to the acquisition of data and manuscript edit and review. LA, FC, AC-R, and LS contributed to the acquisition, analysis, and interpretation of data.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.


In multiple logistic regression analysis adjusted for sex, age, and other potential confounders, Factor 1 scores were associated with a significantly reduced odds ratio (OR) of MetS and all five components. Factor 2 scores were associated with significantly increased prevalence of MetS, obesity, and high blood pressure. Factor 3 scores were significantly associated with lower OR of MetS, high blood pressure, high serum triglycerides and low HDL cholesterol levels. Analysis of nutrient patterns may be useful to assess the overall quality of diet and its association with MetS.The association between nutrient patterns and metabolic syndrome (MetS) has not been examined in a Japanese population. A cross-sectional study was performed on 30,108 participants (aged 35–69 years) in the baseline survey of the Japan Multi-Institutional Collaborative Cohort Study. Dietary intake was assessed using a 46-item food frequency questionnaire. MetS was diagnosed according to the Joint Interim Statement Criteria of 2009, using body mass index instead of waist circumference. Factor analysis was applied to energy-adjusted intake of 21 nutrients, and three nutrient patterns were extracted: Factor 1 ; Factor 2 (fats and fat-soluble vitamins pattern); and Factor 3 (saturated fatty acids, calcium and vitamin B Metabolic syndrome (MetS) is a condition characterized by clustering of abdominal obesity, insulin resistance, high blood pressure, dyslipidemia, and high blood glucose levels, often accompanied by a proinflammatory and prothrombotic state ,2. MetS There are several approaches to investigate the associations of diet with chronic diseases in epidemiologic studies. Dietary pattern analysis has been frequently used because foods and nutrients are consumed as an aggregate, and amounts of foods and nutrients are often highly correlated with each other, making it difficult to investigate the contribution of an individual food or nutrient separately ,9. A priIn the present study, we investigated the associations of nutrient patterns with the prevalence of MetS in individuals who had participated in the baseline survey of the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study.The population for the present study consisted of Japanese men and women aged 35 to 69 years who had participated in the baseline survey of the J-MICC Study. Details of the J-MICC Study have been described in a previous report . BrieflyParticipants from seven study centers that used the same questionnaires and performed blood examinations were included . We excluded participants who had a previous history of ischemic heart disease, stroke or lacked information on previous diseases (No. = 3295), who had missing values for items for the diagnosis of MetS (No. = 7759), or smoking and drinking habits, or physical activity (No. = 1860), or whose total energy intake was extremely high  or low  (No. = 447) . Finally1, B2, C, folate, sodium, energy from alcohol) were computed using a program developed by the Department of Public Health, Nagoya City University School of Medicine. A validation study was performed by comparing the intake of total energy and 26 nutrients estimated using the FFQ and 3 day-weighed diet records. Deattenuated, log-transformed, and energy-adjusted correlation coefficients for intake of total energy and 26 nutrients ranged from 0.10 to 0.86 [1 (0.26) and vitamin A (0.27) [Study subjects were asked to fill out a self-administered questionnaire about dietary habits, current and previous diseases, medication and supplement consumption, physical activity, ways of coping with stress, and smoking and drinking habits. A validated short FFQ was used for dietary evaluation ,22. Part to 0.86 . The corA (0.27) . This FF2). Smoking status was classified as current smoker, past smoker, and never, and drinking status was categorized as current drinker, past drinker, and never. School career was classified as elementary school/junior high school, high school, professional school, junior college/technical college, university/college, graduate school, and others.Physical activity during leisure time was evaluated using a self-administered questionnaire similar to a short format of the International Physical Activity Questionnaire . Total p2 [2; blood pressure ≥130/85 mmHg or treatment for hypertension, serum HDL cholesterol level <40 mg/dL for men and <50 mg/dL for women; serum triglycerides ≥150 mg/dL; and fasting blood glucose level ≥100 mg/dL or treatment for diabetes.MetS was diagnosed according to the Joint Interim Statement Criteria of 2009 [2 . SubjectData on nutrient intake were natural log-transformed and energy-adjusted using the residual method. Factor analysis  with varimax rotation was applied to 21 nutrients after excluding five redundant nutrients. The number of nutrient patterns extracted was determined by eigenvalues, a scree plot and their interpretability.t-test, Wilcoxon’s rank sum test, or chi-square test.Continuous variables are shown as the means ± standard deviations or medians (25th and 75th percentiles), and categorical variables are expressed as the counts and proportions. We examined the differences in participants’ characteristics according to the presence or absence of MetS using the Logistic regression analysis was performed to evaluate the associations of the quartiles of nutrient pattern scores with the prevalence of MetS and its components, after adjustment for potential confounding variables. The first quartile was used as a reference to estimate odds ratios (OR) and their profile likelihood 95% confidence intervals (CI). Model 1 adjusted for age, sex and study center, and model 2 adjusted for variables included in model 1 and total energy intake (quartile), physical activity (quartile), drinking (three categories) and smoking (three categories) habits, and school career (eight categories). Model 3 adjusted for covariates included in model 2 and BMI (quartile). Finally, stratified analyses according to BMI and sex were also performed. Trends across the quartiles were examined by using ordinal categorical variables and a likelihood ratio test. All data analyses were performed using FACTOR, LOGISTIC and other procedures of SAS version 8.2 .n-3 fatty acids, n-6 fatty acids, and vitamin E. Factor 3 was named the “saturated fatty acids, calcium and vitamin B2 pattern” because of a positive correlation with saturated fatty acids, calcium, vitamin B2, and protein, and a negative correlation with carbohydrate. When factor analysis was performed for sex separately, the extracted nutrient patterns were similar  and all five components (p for trend <0.001), obesity, and high blood pressure in Model 2 (p for trend <0.001), high blood pressure, high serum triglycerides, and low serum HDL cholesterol levels , only Factor 1 scores were significantly associated with reduced OR of MetS. In a subgroup with BMI ≥25 kg/m2 , Factor 1 and 3 scores were inversely associated, while Factor 2 scores were positively associated with the prevalence of MetS , calcium was most strongly associated with a reduced OR of MetS , which was considered a reason for the high factor loading of saturated fatty acid on Factor 3. Cross-sectional studies showed inverse associations between calcium intake and MetS in U.S. women [Factor 3 scores were significantly associated with lower OR of MetS and its three components, except for obesity and high blood glucose. Among three nutrients closely correlated with Factor 3 , and 45% and 98% for women (No. = 623), respectively, in the sub-sample of the J-MICC Study in Tokushima Prefecture. Fifth, nutrient patterns may be specific to the study population, and interpretation may be subjective, as is the case for the posteriori dietary pattern analysis. This may make it difficult to generalize the results to other ethnic populations.The strengths of the present study include the large number of study subjects, and use of a validated FFQ. On the other hand, there are also several limitations. First, the number of subjects excluded from the study was large . We compared the characteristics of those who were excluded and not excluded from the final analysis. The most striking difference was the availability of data for the diagnosis of MetS, especially high fasting blood glucose. It is unknown how the results were affected by the exclusion of a relatively large number of study subjects. Second, in cross-sectional studies, the temporal relationship between nutrient intake and onset of MetS is unclear. Third, since nutrient intake was assessed using an FFQ, some degree of measurement error is inevitable. However, only a small number of nutrients showed low validity in our FFQ , and nut2 pattern were associated with a reduced OR of MetS, while a fats and fat-soluble vitamins pattern was associated with an increased OR of MetS. Analysis of nutrient patterns may be useful to assess the overall diet and its association with MetS, and to set dietary guidelines for MetS prevention.In conclusion, the fiber, potassium and vitamins pattern, and saturated fatty acids, calcium and vitamin B