Fibroblast Growth Factor 21 (FGF21), Growth Differentiation Factor 15 (GDF15), and Humanin (HN) are mitochondrial stress-related mitokines, whose role in health and disease is still debated. In this study, we confirmed that their plasma levels are positively correlated with age in healthy subjects. However, when looking at patients with type 2 diabetes (T2D) or Alzheimer’s disease (AD), two age-related diseases sharing a mitochondrial impairment, we found that GDF15 is elevated in T2D but not in AD and represents a risk factor for T2D complications, while FGF21 and HN are lower in AD but not in T2D. Moreover, FGF21 reaches the highest levels in centenarian’ offspring, a model of successful aging. As a whole, these data indicate that (i) the adaptive mitokine response observed in healthy aging is lost in age-related diseases, (ii) a common expression pattern of mitokines does not emerge in T2D and AD, suggesting an unpredicted complexity and disease-specificity, and (iii) FGF21 emerges as a candidate marker of healthy aging.The online version of this article (10.1007/s11357-020-00287-w) contains supplementary material, which is available to authorized users. We have conceptualized that healthy aging, geriatric syndromes (such as frailty), and age-related diseases (ARDs) are part of a gh rate) . In the gh rate) , 3. Mitogh rate) . Mitokingh rate) , 6. Fibrgh rate) –7.Alzheimer’s diseases (AD) and type 2 diabetes (T2D) are among the most common and important ARDs, and both share a metabolic and inflammatory background , 8. AccoGDF15 is a stress response molecule belonging to the transforming growth factor-β (TGF-β) superfamily. GDF15 is produced in response to mitochondrial and inflammatory stressors and is involved in many ARDs, such as cancer, T2D, obesity, and cardiovascular and neurodegenerative diseases. Recent studies demonstrate that GDF15 strongly correlates with aging and is considered a marker of biological age –24. MoreHN is a 24-amino acid mitochondrial DNA-encoded peptide involved in many biological processes associated with inflammatory response, oxidative stress, and apoptosis , 30. HN continuum hypothesis mentioned earlier, we aimed to test the hypothesis that it is possible to identify a sort of trend for mitokine expression from healthy aging to ARDs, as well as similarities and differences between ARDs sharing a common ground of mitochondrial dysfunction such as T2D and AD. To this purpose, we have studied the plasma levels of these three mitokines in a group of > 500 age-matched elderly characterized by different types of aging: healthy controls, centenarians’ offspring (OFF), and patients affected by T2D or AD. Of note, OFF are characterized by a better health status with respect to their age-matched peers and are considered as a reliable example of healthy aging [Within the framework of the hy aging , 36. We A total of 569 subjects in the age range 52–88 years were recruited and divided into five groups, according to their healthy or pathological status: 102 centenarian offspring (OFF), 92 healthy controls (HC), 162 type 2 diabetes (T2D) patients without complications (T2DnC) and 93 T2D patients with complications (T2DC), and 120 patients with Alzheimer’s disease (AD)  was calculated according to CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation based on serum creatinine, age, sex, and ethnicity .APOE genotyping was performed by 7500 Fast Real Time PCR System (Applied Biosystems): DNA samples were genotyped for two APOE single-nucleotide polymorphisms  and relative results defined APOE ε2, ε3, and ε4 alleles. We assigned APOE ε4 status as APOE ε4 negative (ε4−) for APOE ε2/ε3 and APOE ε3/ε3 (non-carriers) and APOE ε4 positive (ε4+) for APOE ε2/ε4, APOE ε3/ε4, or APOE ε4/ε4 .GDF15, FGF21, and HN concentrations were determined in plasma samples by ELISA assay using commercial kits, highly specific for the detection of each human mitokine: R&D for GDF15 (DGD150: intra- and inter-assay coefficient of variation (CV) range: 10.9–1.1% and 4.1–3.0%, respectively; minimum detectable dose 2.0 pg/mL) and FGF21  and CUSABIO for HN , according to the manufacturer’s instructions. In all the samples, GDF15, FGF21, and HN were measured in duplicate, and the mean values were used in the statistical analyses. The standard curves were determined by simultaneously analyzing a dilution series of standard samples. The final data were obtained in a blind set up by the operator. Synergy™ fluorometer  was used to read the absorbance of each plates.y = m + FGF21 + Groupi + FGF21xGroupi, where y = BMI values, FGF21 = covariate effect of FGF21, and Groupi = fixed effect of the ith group . Mediation analysis was performed using model 4 of the PROCESS Macro for SPSS with a bootstrapping procedure involving 10,000 re-samples to generate model estimates and confidence intervals.The data were analyzed with non-parametric tests since they did not follow a normal distribution. In particular, the comparisons among OFF, HC, T2D, and AD patients were performed by using Kruskal-Wallis test, while the comparison between ApoE4+ and ApoE4− AD patients was performed by Mann-Whitney test. The Bonferroni correction was applied. The relationships between each mitokine levels and age were calculated by Spearman rank correlation test and regression analysis. A regression analysis was performed for HC and T2D to evaluate the relationship between BMI and FGF21. The difference between the two regression curves was estimated by the following linear model: R2 statistic.A multinomial logistic regression model using the enter method was constructed to identify factors associated with the presence of T2D. Model fit was assessed using the Hosmer-Lemeshow goodness-of-fit test. The proportion of variance explained by the final model was determined using the Nagelkerke Analysis of covariance (ANCOVA) followed by post-hoc tests for multiple comparisons was used to compare the mean differences in mitokine levels after adjustment for age and sex, and, in case of T2D, glucose-lowering treatment see the “” sectionReceiver operating characteristics (ROC) curves were constructed to assess the discriminatory ability of mitokines in T2D complications. Youden’s index was used to calculate the best cut-off values, where appropriate. Multiple ROC curves were compared using the DeLong method  .Significance was accepted as We measured the levels of GDF15, FGF21, and HN in 569 plasma samples from T2D patients, with (T2DC) or without (T2DnC) complications, AD patients, healthy age-matched controls (HC), and centenarians’ offspring (OFF), see Table p < 0.0001). Moreover, within T2D patients, T2DC showed higher levels with respect to T2DnC (p < 0.01). As far as AD patients, their levels of GDF15 were slightly but significantly higher with respect to OFF (p = 0.012), but not to HC   Fig. . These rPlasma levels of HN were significantly lower in AD patients with respect to all the other groups. No significant difference between healthy subjects (OFF and HC) and T2D patients was observed Fig. .Taken together, these data suggest that these mitokines are modulated in a disease-specific way.As far as the levels of FGF21 in T2D patients, our results are in apparent contrast with literature data showing that T2D patients are characterized by higher levels of FGF21 as compared to controls –44. HoweWe have previously reported that mitokine plasma levels increase with age in healthy people, from young to centenarians, and are correlated with worst hematochemical parameters, including lipid profile . We thenAfter adjusting for age and gender, mitokines in T2D patients were analyzed in relation to the type of treatment, i.e., no treatment (NoT), metformin (Met), sulphonylureas (Sulph), combination of metformin and sulphonylureas (M + S), and insulin (Ins). The circulating levels of GDF15 were significantly higher in patients treated with Met Fig.  with resMoreover, T2D patients often present abnormalities in lipid profile and high risk to develop cardiovascular diseases , 48 and p value: rho = 0.265, p < 0.001).As far as T2D complications, the presence of a complication resulted often associated with a higher level of GDF15 Table , also afSince the levels of the three mitokines were higher in T2D patients with at least one complication, a binary regression analysis was performed to estimate the odds ratio (OR) and 95% confidence interval (CI) of mitokines for predicting the presence of diabetic complications. In addition to mitokines, age, gender, and serum HbA1c were included in this model as covariates, as it is well known that glycemic control is closely associated with the onset of complications , 53. Binp < 0.001) and 0.703 (p < 0.001), respectively. When combined together, GDF15 and HbA1c gave an AUC value of 0.747 (p < 0.001), which is significantly larger than those for the two separated parameters   analysis and compared with that of HbA1c . However, when we performed the same analysis considering the subjects divided by groups, no difference in the levels of the three mitokines was observed between ApoE4 carriers and non-carriers  who are reported to be in a better health status as compared to age-matched peers , 36, andRegarding T2D, all the three mitokines showed interesting associations with the presence of complications and they were related to worsening eGFR. In particular, while confirming literature data indicating that GDF15 increases in T2D , 64, ourConversely, for FGF21, we did not observe any association with T2D. As mentioned in the “p < 0.0001) and glycemia , suggesting that HN could be involved in the maintenance of insulin sensitivity. Taken together, these data suggest that GDF15 and HN are involved in the response to diabetic stress with an opposite regulation, while FGF21 appears to be affected by BMI but not T2D.As far HN and T2D, we did not observe a strong association between them; however, the levels of HN tended to be lower in T2D patients compared to HC, although not significantly after Bonferroni correction. The possible role of HN in T2D is still unclear, and to date, there are still few studies on HN in diabetes. In particular, Voigt and Jelinek showed that the plasma levels of HN are lower in prediabetic patients with impaired fasting glucose compared to a control group , and simConcerning AD, limited data are available on the association between GDF-15 and neurodegenerative diseases. In our study, the levels of GDF15 in AD patients were similar to those of healthy controls. Some studies reported that higher levels of GDF-15  are associated with cognitive impairment and dementia, as well as with decreased gray matter volumes and white matter integrity. All these data suggest that GDF15 could be a possible biomarker for neurodegenerative diseases –72. HoweAs far as FGF21 in AD patients, it has been reported that higher levels of FGF21 have beneficial effects in several pathologies, including neurodegenerative diseases, although the biological function of FGF21 on AD is still largely unclear. To this regard, an in vitro and in vivo study reported that FGF21 attenuated the negative effects of amyloid β-peptide 25–35 on neuronal apoptosis, tau hyperphosphorylation, and oxidative stress in AD-like pathologies . In agreAs far as HN in AD, our results confirm literature data indicating that HN decreases in AD –78. In oAs a whole, our results suggest that GDF15, FGF21, and HN may act synergistically only during physiological aging in the absence of overt diseases. Conversely, when a disease occurs, their expression is modulated differently, even though the considered diseases share a common ground of mitochondrial impairment, like T2D and AD. More studies are needed to clarify the mechanisms underlying this differential modulation.ESM 1(DOCX 17 kb)


The COVID-19 pandemic has impacted community mental health, but the effect on psychiatric admissions is unknown. We investigated factors contributing to acute psychiatric admissions, and whether this changed during the first UK lockdown.A retrospective case-note review study with an exploratory mixed-methods design was used to examine factors in psychiatric admissions following the first UK 2020 lockdown compared to the same time periods in 2019 and 2018.Themes of psychopathology, risk, social stressors, community treatment issues, and physical health concerns were generated. The mean number of codes per case was 6⋅19 (s.d. = 2⋅43), with a mean number of categories per case of 3⋅73, (s.d. = 0⋅98). Changes in routines and isolation were common factors in the study year; accommodation and substance abuse were more prominent in the control year. Relationship stressors featured strongly in both groups. There were significantly more women  = 20⋅80, p < 0⋅00001) and older adults  = 8⋅61, p = 0⋅0033) in the study group than the control. Single people, compared to those in a relationship  = 4.46, p = 0⋅035), and people with affective disorders compared to psychotic disorders  = 5.19, p = 0⋅023), were more likely to have a COVID-19 related admission factor.The COVID-19 pandemic amplified pre-existing psychosocial vulnerabilities with a disproportionate psychiatric admissions impact on the mental health of women, the elderly and those with affective disorders.


Postia placenta and Trametes versicolor, respectively, were cultured in laboratory conditions. In vitro spectral measurements were performed at periodic two week intervals of fungal growth. The study shows structural differences for both species of fungi. The prominent presence of protein amide, carbohydrate and carboxyl bands was of interest. Spectral deconvolution of the infrared broadband around approximately 3300 cm−1 produced peaks at four different wavenumbers. The hydrogen bond energy obtained at the four wavenumbers, from deconvolution, varied from approximately 41 kJ mol−1 to approximately 7 kJ mol−1, indicating the presence of strong and weak forces in microbial cell structure. The hydrogen bond distance, obtained at the deconvoluted wavenumbers, varied between 2.7 Å–2.8 Å, indicating the presence of short and long-distance forces within microbial cells. Microscopic observation showed mycelium colonization, hyphal tip and lateral branching.Decay pathogens follow dissimilar metabolic mechanisms to cause irreversible damage to woody tissues. The objective of this study is to perform inter- and intra-species microbial cell structural comparison using attenuated total reflectance Fourier transform infrared spectroscopy. Representative fungi species, causing brown rot and white rot, namely,  ChemiTrametes versicolor (Fr.) Phil. (Basidiomycota) and brown rot fungus Postia placenta (Fr.) M.J. Larsen & Lombard 1986 (Basidiomycota) were cultured in laboratory conditions. Both species of fungi were cultivated on Norway spruce wood (Picea abies [L.] Karst.). Norway spruce is a widely available tree in northern Europe.Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is used in this study to collect molecular information of microbial cell compositions. ATR-FTIR is being widely used in forensic sciences for surface characterization of biomaterials, polymers and microbes ,8–10. InFagus sylvatica) were exposed to the white rot fungus T. versicolor and was analysed by using ATR-FTIR spectroscopy  Karst.), of dimension: 5 mm × 10 mm × 30 mm, were dried at 103°C for 18 h and dry masses were recorded for each block. The oven-dry specimens were conditioned in room temperature and were set aside for sterilization. Sterilization of the acclimatized specimens was performed at 121°C for 20 min. Statistical analysis was performed to check the variation in mass of wood blocks.Two microbiological culture media were selected to help fungal growth on agar plates. The white rot fungus, . 2.2P. placenta, and the other half (16 nos.) were inoculated with the strains of white rot fungus, T. versicolor. In total, four plates, each containing two wood blocks  inoculated by fungi were evaluated after 2, 4, 6 and 8 weeks of inoculation as followed by previous researchers [Pairs of sterilized wood blocks were placed on each agar plate for each type of fungi as substrates. Half of the agar plates (16 nos.) were inoculated with the strains of brown rot fungus, earchers ,24,26. T. 2.3in vitro, the moisture conditions of the fungal cell were not altered, i.e. living cell characterization was performed simulating normal growth atmosphere. The study assumes that the collection of spectral information in vitro did not alter the microbial cell material compositions within the characterization time frame.Though spectral information was collected −1 (2.5 µm) to 400 cm−1 (25 µm), in an atmosphere with minimalized CO2 and H2O content through purging by a Parker Balston 74-5041 FTIR purge gas generator. With the help of a pair of sterilized tweezers, a thin film of mycelium was placed on the top of the diamond crystal of the ATR-FTIR accessory at ambient conditions. Mechanical pressure from a rotating knob was applied on the top of the specimen to acquire adequate contact with the diamond crystal, i.e. without air pockets at ambient conditions of room temperature and room humidity. Each FTIR spectrum presented is based on a recording of average of 32 scans at a resolution of 4 cm−1. The FTIR spectra presented in this study have not been ATR corrected, neither with respect to penetration depths nor absorbance band shifts, which are dependent on the refractive indices of the sample and the ATR crystal (diamond in this case) and the angle of incident radiation. Because the focus of the study was to compare the information from original ATR-FTIR spectra, no ATR corrections were performed. Besides, the raw ATR-FTIR data, in either transmittance or logarithmic absorbance mode, is usually preferred to perform comparative analysis. It may be noted that errors may get introduced in the ATR corrected spectra for erroneous input of assumed refractive index data. Four replicates of each fungus at each evaluation period were recorded. OMNIC software was used for spectral analysis including spectral derivative and deconvolution. Minitab 16.2.2 was used for statistics.The FTIR material characterization at different periods of fungal growth was performed with a Thermo Nicolet 8700 FTIR spectrometer having a Smart Orbit accessory, i.e. a horizontal ATR accessory (single reflection) with a diamond crystal, wavelength range 4000 cmν0 = standard frequency of the free –OH group at 3650 cm−1, ν = frequency of the bonded –OH group and K = a constant equal to (1/2.625) × 10−2 (kJ mol−1)−1.The hydrogen bond energies from H-N and H-O groups ,28 were −1 regions, the hydrogen bond distances were calculated [ν = ν0 − ν, ν0 = monomeric OH stretching frequency at 3600 cm−1, ν = stretching frequency observed in the infrared (IR) region and R = hydrogen bond distance in Å (1 Å = 10−10 m). OMNIC software was used for spectral analysis. The bands of the nearest frequencies were marked in the enlarged spectra. Between each spectrum, there were differences of 50–100 cm−1. The averages of the energy values and bond distances were thus obtained.To further explore the hydrogen bonds centred on the 3300 cmlculated  from the. 2.4In order to characterize a thin layer of mycelium film a piece of one-sided adhesive tape was lightly touched on the top of fungal culture/colony to collect hyphae on adhesive tape . Scannin. 3T. versicolor and P. placenta, were weighed at two week intervals  in dry mass of wood blocks that were used as substrates, or source of nutrients, to fungi. Therefore, it can be assumed that for the experimental growth conditions and fungi species, similar nutrients were used as substrates. Statistical paired t-test was performed by grouping 32 data of wood mass, for eight replicates at four growth intervals, for each set of fungi. The t-test output , at the 95% confidence limit indicates that there is no statistically significant relationship in the rate of decay loss of wood substrate caused by two different species of fungi.The wooden blocks of Norway spruce were used as nutrient source to fungi. Wood inoculated with the strains of ntervals . Before . 3.1T. versicolor and P. placenta fungi specimens are represented in figures −1 representing the H-bonded hydroxyl (OH) and NH groups, the region around 1636 cm−1 representing the amide bands, and the region around 1030 cm−1 representing the primary-OH from cellulose (CO) [−1 and approximately 3170 cm−1, respectively, are prominent owing to the polypeptide backbone and hydrogen bonding [−1 and a sharper band around 1640 cm−1, which possesses identification challenge owing to the overlapping bands with various organic IR bands [The average FTIR spectra obtained by scanning the cellular structure of the ose (CO) ,32. The  bonding . Note thIR bands . HoweverIR bands ,36. Cons−1 is assigned to amide I for C=O stretching vibrations with contributions from the out-of-phase CN stretching vibration, the CCN deformation and the NH bending of protein [−1 is assigned to amide II for N-H in-plane bending and the CN stretching vibration of protein [−1 for CN stretching and N-H bending with contribution from CO in-plane bending is assigned to amide III [−1 for CH2 bending mode, a band at 1402 cm−1 for C=O symmetric stretching of COO− groups and the band at 1377 cm−1 for C-H bending mode of CH2 [−1–880 cm−1. The band at 1030 cm−1 is assigned to the C-O deformation in primary alcohols as complex bands resulting from C-O and C-O-C stretching vibrations [2 show bands at 1237 cm−1 and 1082 cm−1 for asymmetric stretching and symmetric stretching vibrations, respectively [T. versicolor and P. placenta.In this work, the IR band at 1636 cm protein . The ban protein –39. The  protein . The banmide III . Cell lie of CH2 ,40. The brations . Phosphoectively ,40. ThesT. versicolor. The broad absorbance for the hydrogen-bonded OH group at 3300 cm−1 indicates that the structural components within the fungal cell show high affinity towards hydrogen bonding, especially with water [P. placenta show a similar tendency, i.e. there is a widening of peak in band absorbance centring around 3300 cm−1. There is strong absorbance in the amide regions and at wavenumber 1030 cm−1.−1 for T. versicolor. A steady increase in absorbance of the amide I band at 1636 cm−1 indicates increase in protein content in the fungal cell material from the 2nd week of growth to the 8th week of growth. This may be owing to the reason, that as the fungal colonization increases on the wood substrates, the invading microbes get more nutrient access compared to the starting period because of the process of enzymatic degradation. A strong band, ν(C=C), is observed for the side chain of histidine amino acid, 4-methylimidazole, at 1633 cm−1 [−1 for symmetric stretching vibration, −1, indicating an increase in protein content with growth period. Other researchers have also observed increase in protein content in fungal cell from FTIR studies and concluded that protein increase is related with cell maturity [633 cm−1 . Additioresidues . There amaturity .P. placenta, shows increasing absorbance of the amide I band with increasing period of fungal growth. The absorbance of the band at 1310 cm−1 for amide III, after the 6th week of growth is high compared to the growth patterns of the other three curves. Similarly, the absorbance of the band at 1636 cm−1 for amide I, C=O stretching, for the 4th week of growth is high compared to the growth patterns of the 2nd week growth curve. There are two shoulders at 1700 cm−1 and 1686 cm−1, respectively, which are assigned to the carboxyl group from acids since P. placenta is a known species for secreting an acid sheath to surroundings. The band could also result from the protein amino acid, aspartic acid (C=O), indicating hydrogen bonding to the hydroxyl group [−1 and loss of protein amide II band centring at 1546 cm−1 in the 6th week, which is contrary with the observation of T. versicolor. This indicates that there could be lesser influence of the protein amide N-H molecules. Additionally, this may indicate that the spectra were collected from the hyphal tip where the concentration of protein is less, as was found by other researchers [The expanded IR region in yl group . There iearchers . This al−1 indicates the presence of higher moisture in the cell structure of T. versicolor compared to the cell structure of P. placenta after the 2nd week and 8th week of observations during fungal growth. The spectra from both species of fungi show sharpness in IR bands for fatty acid, amide I and polysaccharide regions.The comparative spectra for the growth of the two types of fungi are shown in . 3.2−1 were performed and plotted graphically as shown in figures 2O and D2O media [2O provide much more reliable data than in D2O media, as the latter changes the protein property owing to exchange of H-D in peptide linkages [2O media [2O, are representations of true O-H bond structure.In order to further investigate the spectral features, second derivatives of the spectral region 1800–900 cm2O media . Howeverlinkages . In addi2O media . Therefo−1, β-sheets near 1637 cm−1, random coil at 1648 cm−1 [Previous researchers have assigned IR bands to the second derivative of receptor protein FTIR spectra as: α- helices at 1653 cm648 cm−1 . Table 2. 3.2.1T. versicolor were prominent. Absorbance bands in the original spectra are shown as negative bands in the second derivative spectra. The splitting of the amide I spectrum shows appearance of a band at 1636 cm−1 and 1644 cm−1 that can be assigned to the ß-sheet and random coils, respectively, of T. versicolor. The protein amino acids absorbance bands for, histidine, ν(C=C), at 1633 cm−1 and salt-bridged arginine, −1, co-appear in the same region [−1 that shifts to lower wavenumber with increasing helix length [−1 that shifts to higher wavenumber with twisting and reduces the splitting between two bands [−1 is assigned to α-helices of the amide III absorption band owing to in-phase combination of CH stretching and NH bending vibrations [−1 band of T. versicolor into two: (i) one at 1036 cm−1 showing presence of carbohydrate C-C bond and (ii) the other at 1020 cm−1 indicating primary alcoholic ν(C-O), both are probably from accumulated cellulose owing to thickening in the cell wall.e region . The α-hx length . The ß-swo bands . At lowebrations . The sec. 3.2.2−1) and amide II bands are shown in P. placenta, in −1, disordered amide I band at 1644 cm−1 and ß sheet at 1625 cm−1 [−1 is often strong [−1, after eight weeks of growth, is relatively stronger than the 1644 cm−1 band absorbance after two weeks of growth. This suggests that the structure of protein might have changed with an increasing period of growth. The antisymmetric band, −1 [−1 [−1 [−1 and 1283 cm−1 are assigned to α-helices and ß-sheet of amide III band owing to in-phase combination of CH stretching and NH bending vibrations [−1, suggesting dominant presence of the primary alcohol C–OH group, which possibly originated from the cellulosic C–OH group. There is a small and sharp band at 1505 cm−1 in the second derivative spectra of P. placenta, indicating the possible presence of lignin; or, the band at 1505 cm−1 could be from the bands of stretching vibrations, ν(CN), δ(CH), δ(NH), of the amino acid tryptophan, that is found at 1509 cm−1 [−1) from the spectra of the P. placenta cell is split into three bands  suggesting the possible presence of the α-helices and ß-sheet structure of cell proteins. The antisymmetric band, νas(COO−), of the protein glutamic acid, is observed in the region 1560–1556 cm−1 [νas(COO−), of the protein aspartic acid, is observed in this band region upon cation chelation [The second derivative of the amide I , (ii) free water (νs stretching), (iii) cluster water not fully coordinated (νas), and (iv) tetrahedral coordinated water (νs stretching) [νas stretching of free water is weak, which leads to the conclusion that most of the water molecules, accessed by ATR crystal, within the microbial cell is hydrogen bonded.The 3600–3050 cmetching) . The int. 3.4−1 region. The hydrogen bond (H-bond) energies for fungal growth at the 2nd, 4th, 6th and 8th weeks are given in −1 and 3390 cm−1, which might have been influenced by the weak signal from free water OH (νs and νas stretching) is low in magnitude (approx. 7.2 kJ mol−1 and approx. 18.5 kJ mol−1). Contrarily, the H-bond at 3229 cm−1 and 3084 cm−1, which might have been influenced by the strong signal from bound/coordinated water OH (νs and νas stretching) is high in magnitude (approx. 30.0 kJ mol−1 and approx. 40.1 kJ mol−1). At high wavenumber, 3554 cm−1, there is a decrease in H-bond energy with growth duration ) that were present in the broadband 3600–3050 cmh period .Table 3t’ test was performed assuming equal mean for the pair of fungal species to evaluate the statistical difference between growth periods  versus absorbance wavenumbers . From p-values as 0.354, 0.043, 0.763 and 0.190, respectively for 3554 cm−1, 3390 cm−1, 3229 cm−1 and 3084 cm−1 wavenumbers. Thus, it can be statistically inferred that H-bond energy at 3390 cm−1, linked with free water (υs stretching [p = 0.043), in IR bands obtained from P. placenta being comparatively higher in magnitude, at four observation periods. There is no statistically significant difference observed for different growth periods in H-bond energies at other wavenumbers. Moreover, from p-values as 0.108, 0.861, 0.474 and 0.900. Therefore, it may be concluded that there is no statistically significant difference between H-bond energies observed individually in the growth period at 2nd, 4th, 6th and 8th weeks for the studied pair of fungi.A paired- ‘. 3.5νas stretching) is longer in dimension than the H-bond linked with bound water (νs and υas stretching) in the cell. Moreover, comparing tables H, kJ mol−1) increases. Other researchers observed that, in a H-bond environment of several protein amino acids and a water molecule, the hydroxyl oxygen is 2.98 Å away from a carbonyl (C=O) oxygen and 2.7 Å from the oxygen atom of the water (H2O) molecule [−1) and stretching of H-bonds (average 2.83 Å) observed at deconvoluted IR band 3554 cm−1 compared to results obtained from other deconvoluted bands. The reported H-bond distance in wood cellulose varies from 2.83 Å at IR band 3567 cm−1 to 2.76 Å at IR band 3252 cm−1 [−1 were 30.35 kJ mol−1 and 2.75 Å, respectively, as observed by other researchers [molecule . The rep252 cm−1 . In woodearchers . In lignearchers . Therefo. 3.6P. placenta. It has been revealed by previous researchers that hyphal tip extension is dominant during the initial stages of the growth process [−1 and amide II band at 1546 cm−1, observed during the 2–8 weeks growth period. Furthermore, the weak H-bond energy at 3390 cm−1, linked with free water (υs stretching), is high from the cell structure of P. placenta which might be linked with imbibed wall pressure and hyphal wall fibril synthesis.Morphology of the mycelial growth was characterized by SEM images.  process . Increas. 4T. versicolor and P. placenta, grown on Norway spruce wood were evaluated by ATR-FTIR spectroscopy at 2nd, 4th, 6th and 8th weeks of growth with the following observations: (i)the statistical analysis shows that there is no significant relationship in decay/mass loss in wood substrate caused by the two species of fungi; (ii)−1 and amide II band at 1546 cm−1; cell lipids show band at 1465 cm−1 for CH2 bending mode; band at 1402 cm−1 for C=O symmetric stretching of COO− groups; band at 1377 cm−1 for C-H bending mode of CH2; 1030 cm−1 is assigned to the C-O deformation in primary alcohol; phospholipids and PO2 show bands at 1237 cm−1 and 1082 cm−1;common IR bands: amide I band at 1636 cm (iii)T. versicolor: protein α-helices at 1655 cm−1; protein random coils at 1644 cm−1; protein ß-sheet at 1636 cm−1; histidine amino acid, 4-methylimidazole, at 1633 cm−1; amino acid arginine symmetric stretching vibration near 1635 cm−1; α-helices of amide III at 1283 cm−1; carbohydrate C-C bond at 1036 cm−1; primary alcoholic ν(C−O) at 1020 cm−1;IR bands assigned to  (iv)P. placenta: protein α-helices at 1654 cm−1; protein random coil at 1644 cm−1; ß sheet at 1625 cm−1; amino acid arginine antisymmetric band region 1695–1652 cm−1; amino acid arginine symmetric band region of 1663–1614 cm−1; protein amino acid lysine asymmetric band region of 1629–1626 cm−1; protein α-helices and ß-sheet structure at 1558 cm−1 - 1542 cm−1; protein glutamic acid antisymmetric band in the region 1560–1556 cm−1; amide III at 1310 cm−1; primary alcohol C–OH group at 1030 cm−1; two shoulders of acid carboxyl group at 1700 cm−1 and 1686 cm−1; amino acid tryptophan stretching vibrations at 1505 cm−1;IR bands assigned to  (v)a steady increase in protein and carbohydrate content in the fungal cell material during the growth period; and (vi)−1 broad OH region shows presence of four bands at 3554 cm−1, 3390 cm−1, 3229 cm−1 and 3084 cm−1 which provides further estimations for the H-bond energy and the H-bond distance. The H-bond energies at the four bands were in the range of 6.8–40.5 kJ mol−1. The H-bond energy at higher wavenumbers is less in magnitude than the bond energy at the lower wavenumbers, indicating stronger interactions at lower wavenumber. The H-bond distances at the four different deconvoluted bands were found to be in decreasing order of magnitude 2.83 Å, 2.79 Å, 2.76 Å and 2.72 Å, respectively, at decreasing wavenumbers. The sequence of the H-bond distance reaffirmed the findings that at lower wavenumber, the interactions are stronger. The microscopic study further confirmed the findings of FTIR spectra by showing the presence of hyphal tip and mycelium.deconvolution of 3600–3050 cmThe consistency of spectral findings that were obtained from the (i) raw data, (ii) 2nd derivative, and the (iii) Fourier deconvolution, shows the efficiency of ATR-FTIR spectroscopy in analysing microbial cell in vivo conditions that confirmed the findings of microscopic images. Further studies may be done to integrate the spectral data, from microbial cells, with the time-related microscopic studies.In this study, cell structures of the two wood decay fungi, namely, Click here for additional data file.


This article was republished on January 25, 2021, to correct errors in the symbols. In the originally published version, the Greek “alpha” symbol (α) appeared incorrectly as either the letter “a” or as a corrupted symbol. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Comprehensive screening for non-alcoholic fatty liver disease (NAFLD) may help prompt clinical management of fatty liver disease. A family history, especially of diabetes, has been little studied as a predictor for NAFLD. We characterized the cross-sectional relationship between a family history of type 2 diabetes (FHT2D) and NAFLD probability in 1185 diabetes-free Apulian  subjects aged > 20 years with overweight or obesity not receiving any drug or supplementation. Clinical data and routine biochemistry were analysed. NAFLD probability was defined using the fatty liver index (FLI). A first-degree FHT2D was assessed by interviewing subjects and assigning a score of 0, 1, or 2 if none, only one, or both parents were affected by type 2 diabetes mellitus (T2DM). Our study population featured most females , and 48.4% (N = 574) of the sample had first-degree FHT2D. After dividing the sample by a FHT2D, we found a higher BMI, Waist Circumference (WC), and diastolic blood pressure shared by FHT2D subjects; they also showed altered key markers of glucose homeostasis, higher triglyceride levels, and worse liver function. FLI scores were significantly lower in subjects without a first-degree FHT2D. After running logistic regression models, a FHT2D was significantly associated with the NAFLD probability, even adjusting for major confounders and stratifying by age (under and over 40 years of age). A FHT2D led to an almost twofold higher probability of NAFLD, regardless of confounding factors . A first-degree FHT2D acts as an independent determinant of NAFLD in excess weight phenotypes, regardless of the age group (younger or older than 40 years). A NAFLD risk assessment within multidimensional screening might be useful in excess weight subjects reporting FHT2D even in the absence of diabetes. Along these lines, the global Burden of Disease epidemiological overview works well as a filter for areas of intervention. Thus, according to the latest register, multimorbidity and polypharmacy affect more than half of all adults, and chronic diseases top the list of health burdens to be faced2. Non-alcoholic fatty liver disease (NAFLD) is currently among the most common metabolic liver disease, consisting of fat accumulation not attributable to secondary causes . The latest reports indicate steadily increasing figures for NAFLD, affecting 20% of the general population and up to 70% of the population with diabetes4. However, the pathological course of NAFLD features a broad spectrum of features ranging from basic steatosis to steatohepatitis (NASH) or fibrosis, leading to cirrhosis and, in the worst cases, hepatocarcinoma. In this ruinous perspective, obese phenotypes with visceral ectopic fat accumulation, i.e., almost 80% of fatty liver phenotypes, carry a higher risk of liver damage5.In this context, liver diseases have been recorded as one of the most alarming epidemiological data among non-communicable diseases in the last decade6; in this regard, we recently reported evidence of a possible bidirectional link whereby HbA1c acts as a collider7. As regards the direct association between exposure to glycaemic alterations and risk of NAFLD, a large body of evidence is provided in the literature, even in diabetes-free subjects8.Beyond higher levels of liver enzymes, the pathophysiological pathways underlying NAFLD involve altered glucose homeostasis9 and is therefore included in many diabetes predictive models10. Of note, this phenomenon is dependent on the hereditary diabetic disease burden, so a FHT2D in both parents is more strongly associated with an impaired residual β-cell function13. Several studies in subjects with obesity demonstrated that a FHT2D accelerates the development of atherosclerosis among diabetes-free subjects. In particular, a FHT2D was found to increase the intima-media thickness of the common carotid artery, a surrogate marker of coronary atherosclerosis14 or the white blood cell count15, and to decrease sensitivity to activated protein C16. Moreover, there is consistent evidence that a FHT2D was associated with endothelial dysfunction17.A further determinant of individual pathological risk trajectories is a family history of diabetes. A family history of type 2 diabetes (FHT2D) is a well-known strong risk factor for the onset of diabetes18.However, to date, little prospective investigation has explored the impact of a family history of diabetes on the risk of chronic disorders including liver disease. The multi-layered significance of the family clinical history does not aid a simple understanding of the underlying pathophysiological pathways, as diabetic familiarity depends on both heritable changes in gene expression and epigenetic driving forces. More than 75 independent genetic loci were found to be associated with type 2 diabetes mellitus (T2DM) in the first genome-wide association study (GWAS), and the epigenetic factor influenced by the surrounding environment also contributes significantly to the pathological endpoint19; in this regard, it is not surprising that the authors found attenuation of this association after adjustment for diet, lifestyle, and adiposity, confirming the important role of these modifiable external factors. Furthermore, a multicenter cross-sectional study of adults with NAFLD diagnosed by abdominal ultrasound reported a FHT2D in 36.5% of participants20; more than 50% of these cases were complicated by overweight or obesity.However, there is some evidence that a FHT2D is associated with higher fasting liver enzyme levels in the adult EPIC-Netherlands cohort without T2DM21. To note, the feature of native homogeneity of the population under study downplays selection bias in our survey, as it assumes a similar predisposition to environmental, lifestyle, and genetic risk factors.To our best knowledge, no study performed in a large southern Italian cohort in Europe has previously investigated whether a FHT2D is able to predict the NAFLD probability, regardless of the presence of diabetes and other confounding variables. The present research study, performed in 1185 Apulian  subjects with an excess weight phenotype but without diabetes, was conducted to examine any relationship between a first-degree FHT2D and NAFLD, independently of major glucose metabolism biomarkers potentially acting as confounding factors associated with NAFLD in excess weight phenotypes2 and receiving no supplements or medication, including oral contraceptives or medicines for osteoporosis. Exclusion criteria were any endocrinological diseases , chronic inflammatory diseases, stable hypertension, angina pectoris, a history of stroke, transient ischaemic attack, atrial fibrillation, heart infarction, congenital heart disease, any major malignancies, kidney or liver failure, inherited thrombocytopenia, HBV or HCV infections, and excess dietary alcohol intake. After excluding subjects not satisfying the inclusion criteria, the final study population consisted of 1185 subjects  aged > 20 years. A summary flowchart of the population screening process is shown in Fig. From January 2018 to December 2020, in total, 3675 patients were recruited at the “Population Health Unit” of the National Institute of Gastroenterology “Saverio De Bellis”, Research Hospital . All data were collected at the baseline examination. Clinical variables were subsequently screened for adequacy against two inclusion criteria, i.e., BMI ≥ 25 kg/m23, suggesting a threshold of 20 g/day in females and 30 g/day in males, those subjects reporting a positive response were excluded from the final study population. Extemporaneous outpatients’ diastolic (DBP) and systolic blood pressure (SBP) was determined in a sitting position after at least a 10-min rest, a minimum of three different times, using an OMRON M6 automatic Blood Pressure monitor. A smoking habit was also investigated as a dichotomous variable (yes/no). Blood samples were drawn at 08.00–09.00 am, after overnight fasting. The blood cell count was determined with a Coulter Hematology analyzer . Fasting plasma glucose (FPG), HbA1c, insulin, total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and liver markers serum levels were assayed. Serum insulin concentrations were measured by radioimmunoassay , and all samples were analyzed in duplicate. Fasting plasma glucose was determined using the glucose oxidase method , while the concentrations of plasma lipids  were quantified by an automated colorimetric method . HbA1c was routinely assayed on a chemical analyzer Architect c8000 (Abbott). Amino transferase and gamma-glutamyl transpeptidase (γGT) were measured with standard routine laboratory methods. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation24. Insulin resistance was assessed using the homeostasis model assessment–insulin resistance (HOMA-IR)25.At baseline, metabolic and routine biochemistry parameters were closely examined in all subjects. A brief interview, including questions about medical history and lifestyle, was conducted by a senior physician. Information about diabetic familiarity was collected by interviewing subjects and assigning a score of 0, 1, or 2 if none, only one, or both parents were affected by type 2 diabetes mellitus (T2DM). Information on daily alcohol consumption was assessed by direct questioning while taking the medical history, as follows: “do you drink at least two glasses of alcohol per day?” for males or “do you drink at least one glass of alcohol per day?” for females. In accordance with the American and European guidelines for daily alcohol consumption2) and classified according to World Health Organization criteria for normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), grade I obesity (30.0–34.9 kg/m2), grade II obesity (35.0–39.9 kg/m2), and grade III obesity (≥ 40.0 kg/m2)26. Waist circumference (WC) was measured at the narrowest part of the abdomen or in the area between the tenth rib and the iliac crest (minimum circumference).Two qualified nutritionists , trained for equivalent measuring performances, carried out clinical procedures. All anthropometric measurements were taken with participants dressed in lightweight clothing and without shoes. Variables were all collected simultaneously at 7.00–10.00 am, after overnight fasting. Height was measured to the nearest 0.5 cm using a wall-mounted stadiometer . Bodyweight was determined to the nearest 0.1 kg using a calibrated balance beam scale . BMI was calculated by dividing body weight (Kg) by the square of height  + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × WC—15.745)/(1 + e 0.953 × loge (TG) + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × WC—15.745) × 100. Subjects with FLI < 30 are classified as at low risk of NAFLD, and those with FLI ≥ 60 at high risk.The FLI, a modelling algorithm including BMI, WC, triglycerides, and γGT28.We performed descriptive analysis of baseline variables, expressed as mean ± Standard Deviation (SD), and proportion (%) for the frequency of categorical variables. First the sample was divided into two categories, according to a positive or negative FHT2D, and subsequently we performed the same descriptive analysis on three categories, to appreciate the differences between subjects with only one diabetic parent or both. The normality of distribution was assessed for each variable using Shapiro’s test. A non-parametric approach was used to assess statistically significant differences between groups for all collected variables. The distribution of each variable was reported as mean ± SD for continuous variables, and as proportion (%) to facilitate comparison between our study and others, as these are more generalizable and reliable measures than median and range. Mann Whitney sum rank test was used to assess statistically significant differences between groups for continuous variables, and Chi square test for categorical variables. p-values less than or equal to 0.05 were considered statistically significant, with 95% confidence intervals. A Spearman’s correlation matrix was built for all continuous biochemical and anthropometric variables to check for interrelated variables and so avoid collinearity effects in the model (Supplementary Table p < 0.01), WC (p < 0.01), and extemporaneous diastolic BP (p = 0.03) were found to be clinical features common to subjects with at least one diabetic parent. These subjects also exhibited poorer levels of major glycaemic metabolism parameters, i.e., FBG, insulin, HOMA index, and HbA1c (p < 0.01). As to lipid metabolism, the same subjects presented significantly higher levels of triglycerides (p < 0.01), but no significant differences compared to the FHT2D group for total- (p = 0.49), HDL- (p = 0.26), and LDL-cholesterol (p = 0.92) levels. When considering liver function parameters, however, the FHT2D group had significantly higher mean levels of circulating AST (p = 0.04), ALT (p < 0.01), and gamma-GT (p < 0.01). Thus, the FLI score was significantly higher in subjects who had at least one parent affected by T2DM (p < 0.01), and a pathological value (FLI ≥ 60) was found to be highly prevalent in the same subjects group  as compared with those without a FHT2D (p < 0.01). Table The examined population N = 1185) was dominated by females ; mean age was 47 years, ranging from 20 to 74. A comparative descriptive analysis of the sample by a first-degree FHT2D  is shown in Table 185 was dTable The present study examined a large population of diabetes-free subjects with overweight or obesity, providing evidence of a close positive cross-sectional relationship between a FHT2D and the NAFLD probability, regardless of age group (younger or older than 40 years) (Table 8. Our study fits into this context by further adding one novel aspect, as we illustrate that NAFLD risk may also be explained by FHT2D in the absence of diabetes and any pharmacological therapy, based on a large cohort analysed here. Furthermore, the analysis was performed within a homogeneous cohort from southern Italy in Europe that shares similar characteristics.A body of evidence has already shown how in subjects with NAFLD and a pattern of blood chemistry consisting of both elevated hepatic and glycaemic biomarkers can be predictive of diabetes, and how high HbA1c levels are associated with NAFLD in non-diabetic phenotypes19. This information may be handy in the clinical setting; indeed, while a family history of T2DM was already acknowledged to be a major risk factor for the onset of diabetes10 as well as in speeding up atherosclerotic events in diabetes-free subjects15, we now add the unfavourable new finding of an increased probability of developing NAFLD. Practically speaking, it will be helpful to stress the importance for healthcare clinicians of possibly including a thorough NAFLD risk assessment within a multidimensional screening program for excess weight subjects reporting a FHT2D.This finding, obtained using the FLI, a scoring algorithm based on BMI, WC, and serum γGT and triglycerides levels, appears to be in line with a previous report showing that a family history of T2DM was associated with higher fasting liver enzyme levels in a general diabetes-free population7, thus reinforcing the importance of investigating a FHT2D when caring for individuals with overweight or obesity and free from any drug therapy. Indeed, among these major confounders considered, there is evidence that genetic polymorphisms, sex hormones, and dysmetabolic traits work synergistically well in delineating a female prevalence of NAFLD29. Smoking was also considered in corroborating our findings, consistent with evidence defining smoking habit among major risk factors associated with NAFLD and the most recent metrics indicating approximately 30–40% prevalence of NAFLD among smokers30. It should be noted as well that the association was maintained even after adjustment for age, though the prevalence of NAFLD is known to be negatively affected by aging31; this aspect further strengthens the main association, stressing FHT2D as a matter of consideration.Consistent with this preliminary result, we found impaired glucose metabolism biomarkers, i.e., FBG, insulin, HOMA-IR, and HbA1c, to be clinical features common to FHT2D subjects. Aligned with the presence of insulin resistance, significantly higher triglycerides values were found in the same group. Most interestingly, the association between a FHT2D and the NAFLD probability in this study was corroborated independently of age, sex, and all factors well-known to be related to NAFLD Table  such as Notably, further analysis of our study population showed that a dual FHT2D, i.e., both parents with T2DM, did not further increase the likelihood of NAFLD, suggesting that, in some way, lack of a FHT2D might offer some safety against the development of steatosis by protecting against insulin resistance and impaired glucose metabolism.32.Pending further investigation to explain the possible mechanisms underlying our main finding, preventive intervention to tackle the risk of NAFLD is key when examining FHT2D subjects. By targeting familiarity, and being aware that both genetic and environmental factors can weigh on it, acting on the lifestyle front is clearly a good idea. Among preventive strategies, recent longitudinal data are encouraging regarding the efficacy of lifestyle interventions. Specifically, Zhu and colleagues demonstrated that patients with a family history of T2DM and metabolic syndrome benefited more significantly from lifestyle interventions in terms of insulin resistance than those without a FHT2D, regardless of body weight changes, over a 2-year follow-up29. An important limitation is the lack of information on parental lifestyle, assuming that the pattern of association is not only a genetic but also an epigenetic mechanism. Therefore, it would have been useful to investigate parental behavioral and social factors, as well as diet habits. The main strength of this study is that it included only individuals not taking medications, thus avoiding any possible interference with biomarkers testing and survey results.Some study limitations must be considered. Because of the cross-sectional design, we cannot appreciate the temporal nature of the associations, so prospective studies are needed to clarify any causal relationship. Moreover, NAFLD was estimated using a probability scoring tool rather than validated imaging techniques for the diagnosis of NAFLD, although this scoring is still the only guideline-recommended tool for evaluating liver steatosis when imaging and biopsy are not availableWe conclude that a first-degree FHT2D acts as an independent determinant of the NAFLD probability in excess weight phenotypes, markedly worsening the adverse trajectories of overall health status that are commonly attributed to impacts on glucose metabolism. As a consequence, the inclusion of an accurate NAFLD risk assessment within multidimensional screening for overweight individuals reporting a FHT2D becomes a crucial point whose importance needs to be highlighted.Supplementary Table S1.


In older and multimorbid patients, chronic conditions may affect the prognostic validity of computed tomography (CT) findings in COVID-19. This study aims at assessing to which extent CT findings have prognostic implications in COVID-19 older patients. Hospitalized COVID-19 patients aged 60 years or more enrolled in the multicenter, observational and longitudinal GeroCovid study who underwent chest CT were included. Patients were stratified by tertiles of age and pneumonia severity to compare CT findings. Hierarchical clustering based on CT findings was performed to identify CT-related classificatory constructs, if any. The hazard ratio (HR) of mortality was calculated for individual CT findings and for clusters, after adjusting for potential confounders.380 hospitalized COVID-19 patients, with a mean age of 78 (SD:9) years, underwent chest CT scan. Ground glass opacity (GGO), consolidation, and pleural effusion were the three most common CT findings, with GGO prevalence decreasing from younger to older patients and pleural effusion increasing. More severe the pneumonia more prevalent were GGO, consolidation and pleural effusion. HR of mortality was 1.94 (95%CI 1.24–3.06) for pleural effusion and 13 (95%CI 6.41–27) for cluster with a low prevalence of GGO and a high prevalence of pleural effusion (“LH”), respectively. Out of the three CT based clusters, “LH” was the only independent predictor in the multivariable model.Pleural effusion qualifies as a distinctive prognostic marker in older COVID-19 patients. Research is needed to verify whether pleural effusion reflects COVID-19 severity or a coexisting chronic condition making the patient at special risk of death.ClinicalTrials.gov: NCT04379440The online version contains supplementary material available at 10.1186/s12877-022-02837-7. COVID-19 qualifies as a systemic disease with a great variety of symptoms and damaged organs. However, it basically remains a respiratory disease in the vast majority of patients. Indeed, respiratory symptoms and gas exchange indexes have primary classificatory and prognostic implications. CT scan findings have also been repeatedly reported to contribute to the diagnosis and prognostic classification of COVID-19 , 3. CT aThis bulk of evidence clearly states that CT scan plays a primary role in diagnosing and monitoring COVID-19. However, the possible confounding effect of conditions such as chronic obstructive pulmonary disease (COPD) or congestive heart failure (CHF) in the interpretation of CT findings has not been tested so far in older and multimorbid patients. Furthermore, combined and not isolated CT findings might play a major classificatory and prognostic role. Thus, assessing clustered rather than individual CT findings might improve our understanding of CT findings’ clinical meaning. These two topics are the object of the present study. Based on the GeroCovid data, we will provide an estimation of the real-life CT findings’ contribution to the prognostic stratification in older and multimorbid COVID-19 inpatients.st, 2020. The enrolment of new cases ended on December 31st, 2020, and the follow-up will be closed on June 30th, 2021. Overall, 66 investigational sites are actively participating. The final endpoint of GeroCovid is to provide high-quality and comprehensive data, which will help optimize COVID-19 prevention and management of patients > 60 years. GeroCovid conforms to the principles outlined in the Declaration of Helsinki, it was registered at ClinicalTrials.gov (NCT04379440) and the participation of each centre was authorized by the corresponding local Ethical Committee [GeroCovid Observational is a multi-purpose and multicenter initiative promoted by the Italian Society of Gerontology and Geriatrics (SIGG) in collaboration with Bluecompanion that aims at investigating the impact of SARS-CoV-2 pandemics in older patients in different settings of care. The objectives of the project are setting specific. In the GeroCovid acute wards cohort, we purposed to assess COVID-19 clinical presentation and course and identify the prognostic factors of the disease in older people. Data are collected retrospectively and prospectively in an e-Registry since March 1st, 2020 to December 31st, 2020 were retrospectively studied, without any exclusion criteria. All the patients received routine CT scan according to the local institutional protocol, and the CT findings retrieved from the radiological reports were utilized for the final analysis. The number of pulmonary lobes involved was used as a severity score. Demographics, comorbidity, clinical and laboratory data were collected for all patients, including a comprehensive geriatric assessment (CGA). The presence of frailty was also evaluated according to modified Fried criteria [2/FIO2 ratio [2 indicated in the swivel connector was utilized) [Inpatients aged 60 or older who underwent chest CT examination within 48 h from hospitalization and positive reverse transcriptase- polymerase chain reaction test (RT-PCR) for SARS-CoV-2 from March 1criteria . Particicriteria , which hcriteria –14, and:O2 ratio   . PrimaryFor the purposes of this analysis, patients who did not undergo chest CT or did not in time were excluded see . DescripIn order to detect potential classificatory and prognostic constructs inaccessible to an age-based classification of the patients, hierarchical clustering was performed on CT scan data using an agglomerative nesting algorithm (i.e. AGNES). Ward’s method was used as a linkage criterion. The result of clustering was represented using a dendrogram. Five clusters were identified according to the dendrogram and their radiological, clinical, and laboratory characteristics were compared as previously described. The risk of mortality was evaluated using a Kaplan Meier estimator. After checking the proportional hazards assumption using Schöenfeld residuals, the hazard ratio (HR) and 95% confidence interval (95%CI) of mortality was calculated for each CT scan finding and cluster, using the cluster with the lowest risk as a reference. Univariable and multivariable models were performed, the latter being adjusted for age, sex, BMI, frailty, and those diseases with a well-known impact on the CT scan and outcome, i.e. CHF, COPD, and diabetes. Results are represented using a forest plot. All statistics were performed using R version 4.0.2  using the packages “cluster”, “dendextend”, “survival” and “surviminer”.2/FiO2 ratio (mean:250 vs 271), higher prevalence of cognitive impairment (22% vs 13%), shorter hospital stay (median days:3 vs 8) and greater mortality (31% vs 25%)  years, 56% were males, and three quarters were living at home before hospital admission. At baseline, according to pneumonia severity 47% was treated with Oxygen by mask or nasal prongs, 14% with HF/NIV and 5% was intubated and mechanically ventilated. GGO prevalence at CT scan was 85%, consolidation 41%, pleural effusion 23%, pulmonary nodules 12%, and bronchiectasis 9%; all other CT findings had a prevalence of 5% or lower. Images of the three most common CT-findings are reported in 25%) see .Fig. 2Ha2/FIO2, no patient in Group 3 was intubated and mechanically ventilated. The prevalence of GGO progressively decreased from Group 1 to Group 3, while that of pleural effusion, cardiomegaly, and pericardial effusion increased. Of note, there was no difference in the prevalence of pleural effusion , cardiomegaly  and pericardial effusion  between participants with and without chronic heart failure. Furthermore, no difference was found for consolidation and other considered findings. Participants with consolidation did not differed from those without in terms of C-reactive protein – 245 mg/l versus 271 mg/l, P-value 0.58 – and procalcitonin – 10 ng/ml versus 11 ng/ml, P-value 0.47 –, laboratory tests suggestive of sepsis. More details about the study population are reported in Table Group 1 (60–74 years), Group 2 (75–83 years) and Group 3 (≥ 84 years) were composed by 139, 116 and 125 patients, respectively. The prevalence of men significantly decreased from Group 1 to Groups 3, as well as the percentage of individuals living at home, while the percentage of underweight/malnourished individuals increased. Mortality was significantly higher in Group 3 and, although no difference was found among groups in PaOCT scan-based clustering identified five clusters see Fig.  with theCluster 1 showed a low prevalence of both GGO and pleural effusion (“LL”), Cluster 2 a high prevalence of both the previous findings (“HH”), Cluster 3 a high prevalence of GGO and intermediate prevalence of pleural effusion (“HI”), Cluster 4 a low prevalence of GGO and high of pleural effusion (“LH”) and Cluster 5 a high prevalence of GGO and low of pleural effusion (“HL”). Clusters were similar in terms of consolidation, except for Cluster 4 (“LH”) showing a low prevalence. More details on CT-scan findings by clusters are reported in 2/FIO2 and the outcome. Cluster 1 (“LL”) had a PaO2/FIO2 > 300, suggesting a less severe respiratory impairment, which is confirmed by the infrequent use of HF/NIV, Cluster 2 (“HH”), 3 (“HI”) and 5 (“HL”), sharing a high prevalence of GGO, had a PaO2/FiO2 of around 250, with more severe pneumonia frequently treated with HF/NIV or intubation, and Cluster 4 (“LH”) had the lowest PaO2/FIO2, at the threshold for acute respiratory distress syndrome, with the most severe disease. The last cluster showed significantly higher mortality, with Cluster 1 (“LL”) and Cluster 5 (“HL”) having the lowest values. A comparison of clinical and laboratory characteristics between different clusters can be found in Table Furthermore, clusters differed in PaOPleural effusion was the only single CT finding associated with the outcome . Likewise, Cluster 2 (“HH”), Cluster 3 (“HI”), and Cluster 4 (“LH”) showed an increased HR of death. After adjusting for confounders, neither CT scan findings alone nor clusters, except Cluster 4 (“LH”), were significantly associated with the outcome  revealed a better outcome compared to the others. These two clusters presented the lowest prevalence of pleural effusion, yet additional factors might have contributed to a higher survival rate. For example, the majority of patients in “LL” and “HL” clusters were living at home, indirectly suggesting lower disability degree which may have impacted survival. Furthermore, Kaplan Meier curves highlight that while “HL” has a constant mortality rate over the first month, “LL” mortality is steeper in the first week and then declines. Interestingly, even though “HI” and “HL” clusters showed the same median age, they shaped two different phenotypes of COVID-19 patients; older persons in the “HL” cluster were less frequently obese (14% vs 21%) compared to the “HI” cluster. These characteristics might have played a role in mortality specifically in the first period of hospitalization, when most “HI” patients died, compared to “HL” cluster that showed a longer length of stay and a lower mortality.2/FIO2 ratio and the higher mortality risk, resulting independently associated with death even after adjustment for comorbidities. Noteworthy, “LH” patients, alike patients in the “HI” cluster, showed a steep mortality curve, that is a very early mortality.Confirming the important meaning of pleural effusion, the “LH” cluster showed the lowest PaOThis study has several strengths. First, it is one of the largest studies of hospitalized geriatric patients with COVID-19 focusing on CT scan findings, with more than 100 patients aged more than 83 years old. Second, data are representative of real-life. Finally, to the best of our knowledge, this is the first study evaluating the prognostic impact of CT scan findings, alone or grouped in clusters, in old hospitalized patients with COVID-19 adjusting by age, frailty and chronic comorbidities. However, there are some limitations. First, due to variability in practice, many patients did not undergo CT scan. As a fact, missing data might have broadened the generalizability of the current investigation. Nonetheless, both cohorts shared in the majority the same “mild ARDS” category as defined in the Berlin Definition  and no dThis study advances our knowledge about the prevalence and prognostic implications of CT findings in older COVID-19 patients. It discloses a changing prevalence of CT findings with age and allows reconsider the prognostic role of these findings at variance with adult populations, emphasizing the negative prognostic implications of pleural effusion.Additional file 1: Summary of the IRB approvals.Additional file 2:e-Figure 1. CONSORT flowchart diagram. e-Figure 2. Common CT-scan findings in older patients with COVID-19 pneumonia. e-Table 1. Characteristics of the GeroCovid population included (n:380) and excluded (n:820) from the analysis. e-Table2. Ground glass opacities distribution stratified by tertiles of age. e-Table 3. Distribution of CT-scan findings by clustersm.


Increased mobility and human interactions in the Mediterranean region during the eighth through fifth centuries BCE resulted in heterogeneous communities held together by political and cultural affiliations, periodically engaged in military conflict. Ancient historians write of alliances that aided the Greek Sicilian colony Himera in victory against a Carthaginian army of hired foreign mercenaries in 480 BCE, and the demise of Himera when it fought Carthage again in 409 BCE, this time unaided. Archaeological human remains from the Battles of Himera provide unique opportunities to test early written history by geochemically assessing the geographic origins of ancient Greek fighting forces. We report strontium and oxygen isotope ratios of tooth enamel from 62 Greek soldiers to evaluate the historically-based hypothesis that a coalition of Greek allies saved Himera in 480 BCE, but not in 409 BCE. Among the burials of 480 BCE, approximately two-thirds of the individuals are non-local, whereas among the burials of 409 BCE, only one-quarter are non-local, in support of historical accounts. Although historical accounts specifically mention Sicilian Greek allies aiding Himera, isotopic values of many of the 480 BCE non-locals are consistent with geographic regions beyond Sicily, suggesting Greek tyrants hired foreign mercenaries from more distant places. We describe how the presence of mercenary soldiers confronts prevailing interpretations of traditional Greek values and society. Greek fighting forces reflect the interconnectedness and heterogeneity of communities of the time, rather than culturally similar groups of neighbors fighting for a common cause, unified by “Greekness,” as promoted in ancient texts. Antagonistic interactions are not just a reflection of people’s perceptions of each other and themselves, but also a stimulus for the formation of new ethnic identities and political forms, bringing some groups together while cleaving others Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Review of “Isotopic Evidence for Geographic Heterogeneity in Ancient Greek Military Forces” by Reinberger et al. for PLOS OneThe scientific merits of this paper include the investigation of geographic origins in individuals buried after two Greek battles in the Greek Sicilian colony of Himera using strontium and oxygen isotopes. The dataset is a welcome contribution to the use of isotopic data to infer geographic origins. I think it is particularly important to have examples like this one, where the authors successfully incorporate Greek historical documents with bioarchaeological data in an appropriately contextualized bioarchaeological project.The research questions, background sections, sample preparation and analysis are appropriate and the interpretations are reasonable and supported by the data. I only have minor suggestions to improve the paper. For example, the convention is usually to have a minimum of three sentences in each paragraph, and following Coplen, the delta symbol should be italicized throughout. Finally, given the accuracy and precision of the oxygen isotope data from the analyzed standards, the data should only be reported to the first decimal place, not the second.Overall, I feel that the questions and methods addressed in this manuscript are beneficial to the body of literature on isotopic analyses in bioarchaeology. This manuscript was very interesting to read and I am happy recommend the manuscript for publication.Reviewer #2: Overall, this is a very good study using scientific methods to test "historical" information. Very few strontium isotope analyses have been done yet in this area, however oxygen isotope values are available for many archaeological sites in Italy. One example for Sicily, for Greek site in Syracuse, is Tanasi et al. 2017 in Science and Technology of Archaeological Research 3(2): 466-477.Some minor questions just to elaborate on:p. 3, line 58: was The Histories definitely written in 440 BCE, or “about” then?p. 7, lines 150-151: here it says “revealed nine mass graves...” but then in the next sentence “Seven graves...and one.” So is it 8 or 9 in total?p. 9, lines 181-182: Why are “shales and granites” older rocks, and “basalt” a younger rock? Doesn’t this specifically depend on the particular geographic location? The citations provided are not for Sicily or this area in particular.p. 11, line 232-234: So of 132 total individuals, 70 didn’t have any teeth? p. 13 - Table 1: I don’t think it is appropriate to have six decimal places for the Sr isotope ratio, since the values obtained on the standard NBS 987 have std in the 5th decimal place .p. 16, line 306: so you decided to use regular acetic acid rather than buffered, when the latter has been shown to be more consistent between labs and separate from particle size. Any particular reason?p. 16, lines 313-315: was a different mass spec used for the water samples, or just a separate input to the same mass spec as the tooth samples?Minor corrections:p. 4, line 79: add space before “Greekness”p. 5, line 108: should be a period after “Fig”p. 7, line 146: need space after period and before “The”p. 8, line 174: plural is “radii”, not “radiuses”p. 9, line 190 & 192: should be a period after “Fig”p. 9, line 192: Sentence could have better grammar, e.g. “..rocks are associated with the Mt. Etna...”p. 10, line 202: add space bore “87Sr/86Sr”p. 10, line 208: spell out “Fig” since it’s part of the sentencep. 10, line 210: remove the underline under the period and space before “(52)”p. 10, lines 219-224: add space before “mm” and before “C”, these are abbreviations for separate words p. 11, line 25: samep. 11, line 242: change tense of “analyze” to “analyzed”p. 12, line 246: samep. 15, lines 285, 290, 301: add space before “C”p. 15, line 290: remove space in “2 N” to be consistent with what you have elsewhere on the same pagep. 15, line 298: add spaces in both cases where you have “2ml”p. 16, line 303: edit wording so you don’t have “following using”p. 16, line 311: make the sentence better by starting “The mean d18O of the Fisher...”p. 16, line 312: remove the second decimal place from the std dev “0.12"p. 16, lines 310, 315, 319, 321, 322: the “v” in “vPDB” and “vSMOW should be capitalizedp 17, line 337: ditto p. 17, line 334: shorten to 5 decimal places the Sr rangep. 17, line 341: delete “calculated” since it is in the next line of the sentencep. 18, Table 3: change d18O isotope values to one decimal place; capitalize the “v” in column captionp. 18, Table 3: change Sr decimal places to 5p. 21, lines 349-350: be consistent in hyphen size and space before/afterp. 21, lines 349-350 & 356-357: be consistent and use 5 decimal places (not 4 or 6)p. 22, line 30: again you have just 4 decimal places, is that particularly intentional?p. 23, line 376: add something like “The dozen...” at the beginning of the sentence to reinform the reader how many individuals the data are based onp. 23, line 377: you have average of 6 decimal places, std only 5p. 23, line 380: add space between “d18O” and “value”, and make “18" superscriptp. 23, line 384: add space againp. 23, line 392: after “values” add “which” and remove “s” from “supports” so it reads “...values which support literary...”p. 23, line 394: reword beginning of sentence, e.g. to “This study provides evidence, however, for..”p. 24, line 420: are “Sicels” the same as “Sikels”? Check if you mention either before this pagep. 25, line 443: change “offer” to past tense (offered)p. 26, line 462: add space after period, before “Foreign”p. 26, line 468: change semicolon to comma after “Stamer”p. 28, ref. 1: remove the “Available from: ...”p. 29, line 550: capitalize second initial for Ezzo, uncapitalize “Of” in journal namep. 29, line 564: remove space before colonp. 30, line 572: remove the “Available from: ...”p. 30, line 585: remove the “Available from: ...”p. 30, lines 609-10: capitalization of article title inconsistent with others, check the style expected.p. 31, line 633: remove space after hyphen so that it reads “Look-Up”p. 31, lines 635-7: remove the “Available from: ...”Figure 3: the precision of the O isotope ranges should be consistent with decimal places, while given the precision of the measurements no more than 2 decimal places. Also the lower case “v”**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  1 Mar 2021The authors are appreciative of the thorough comments by the reviewers. The following document includes the original comments by the reviewers and editor followed by corresponding responses by the author. Additionally, during revisions, the authors discovered a discrepancy between the text and Figure 4 in the baseline Sr range for Agrigento. We have fixed the range in Figure 4 to be consistent with the calculated range in the text. Finally, we included a citation in the discussion section to a new publication about 87Sr/86Sr variation in the Mediterranean region: Leppard TP, Esposito C, Esposito M. The bioarchaeology of migration in the ancient Mediterranean: Meta-analysis of radiogenic (87Sr/86Sr ) isotope ratios. Journal of Mediterranean Archaeology. 2020;2: 211–41.Reviewer Comments and Author ResponsesReviewer 1:The scientific merits of this paper include the investigation of geographic origins in individuals buried after two Greek battles in the Greek Sicilian colony of Himera using strontium and oxygen isotopes. The dataset is a welcome contribution to the use of isotopic data to infer geographic origins. I think it is particularly important to have examples like this one, where the authors successfully incorporate Greek historical documents with bioarchaeological data in an appropriately contextualized bioarchaeological project.The research questions, background sections, sample preparation and analysis are appropriate and the interpretations are reasonable and supported by the data. I only have minor suggestions to improve the paper. For example, the convention is usually to have a minimum of three sentences in each paragraph, and following Coplen, the delta symbol should be italicized throughout (Author response: DONE). Finally, given the accuracy and precision of the oxygen isotope data from the analyzed standards, the data should only be reported to the first decimal place, not the second (Author response: DONE).Overall, I feel that the questions and methods addressed in this manuscript are beneficial to the body of literature on isotopic analyses in bioarchaeology. This manuscript was very interesting to read and I am happy recommend the manuscript for publication. Reviewer 2Overall, this is a very good study using scientific methods to test "historical" information. Very few strontium isotope analyses have been done yet in this area, however oxygen isotope values are available for many archaeological sites in Italy. One example for Sicily, for Greek site in Syracuse, is Tanasi et al. 2017 in Science and Technology of Archaeological Research 3(2): 466-477.Some minor questions just to elaborate on:p. 3, line 58: was The Histories definitely written in 440 BCE, or “about” then? p. 7, lines 150-151: here it says “revealed nine mass graves...” but then in the next sentence “Seven graves...and one.” So is it 8 or 9 in total? p. 9, lines 181-182: Why are “shales and granites” older rocks, and “basalt” a younger rock? Doesn’t this specifically depend on the particular geographic location? The citations provided are not for Sicily or this area in particular. p. 11, line 232-234: So of 132 total individuals, 70 didn’t have any teeth?   p. 13 - Table 1: I don’t think it is appropriate to have six decimal places for the Sr isotope ratio, since the values obtained on the standard NBS 987 have std in the 5th decimal place . p. 16, line 306: so you decided to use regular acetic acid rather than buffered, when the latter has been shown to be more consistent between labs and separate from particle size. Any particular reason?.)p. 16, lines 313-315: was a different mass spec used for the water samples, or just a separate input to the same mass spec as the tooth samples?(Author response: A different mass spec was used for the water samples. Both mass specs were Thermo Delta V plus IRMS. The water samples are routed from the TC-EA to the Delta V via a Conflo III interface.)Minor corrections:p. 4, line 79: add space before “Greekness” (Author response: DONE)p. 5, line 108: should be a period after “Fig” p. 7, line 146: need space after period and before “The” (Author response: DONE)p. 8, line 174: plural is “radii”, not “radiuses” (Author response: DONE)p. 9, line 190 & 192: should be a period after “Fig” p. 9, line 192: Sentence could have better grammar, e.g. “..rocks are associated with the Mt. Etna...” (AUTHOR RESPONSE: DONE)p. 10, line 202: add space bore “87Sr/86Sr” (Author response: DONE)p. 10, line 208: spell out “Fig” since it’s part of the sentence (Author response: DONE)p. 10, line 210: remove the underline under the period and space before “(52)” (Author response: DONE)p. 10, lines 219-224: add space before “mm” and before “C”, these are abbreviations for separate words  (Author response: DONE)p. 11, line 25: same (Author response: DONE)p. 11, line 242: change tense of “analyze” to “analyzed” (Author response: DONE)p. 12, line 246: same (Author response: DONE)p. 15, lines 285, 290, 301: add space before “C” (Author response: DONE)p. 15, line 290: remove space in “2 N” to be consistent with what you have elsewhere on the same page (Author response: DONE)p. 15, line 298: add spaces in both cases where you have “2ml” (Author response: DONE)p. 16, line 303: edit wording so you don’t have “following using” (Author response: DONE)p. 16, line 311: make the sentence better by starting “The mean d18O of the Fisher...” (Author response: DONE)p. 16, line 312: remove the second decimal place from the std dev “0.12" (Author response: DONE)p. 16, lines 310, 315, 319, 321, 322: the “v” in “vPDB” and “vSMOW should be capitalized (Author response: DONE)p 17, line 337: ditto  (Author response: DONE)p. 17, line 334: shorten to 5 decimal places the Sr range (Author response: DONE)p. 17, line 341: delete “calculated” since it is in the next line of the sentence (Author response: DONE)p. 18, Table 3: change d18O isotope values to one decimal place; capitalize the “v” in column caption (Author response: DONE)p. 18, Table 3: change Sr decimal places to 5 (Author response: DONE)p. 21, lines 349-350: be consistent in hyphen size and space before/after (Author response: DONE)p. 21, lines 349-350 & 356-357: be consistent and use 5 decimal places (not 4 or 6) (Author response: DONE)p. 22, line 30: again you have just 4 decimal places, is that particularly intentional? p. 23, line 376: add something like “The dozen...” at the beginning of the sentence to reinform the reader how many individuals the data are based on (Author response: DONE)p. 23, line 377: you have average of 6 decimal places, std only 5 (Author response: DONE)p. 23, line 380: add space between “d18O” and “value”, and make “18" superscript (Author response: DONE)p. 23, line 384: add space again (Author response: DONE)p. 23, line 392: after “values” add “which” and remove “s” from “supports” so it reads “...values which support literary...” (Author response: DONE)p. 23, line 394: reword beginning of sentence, e.g. to “This study provides evidence, however, for..” (Author response: DONE)p. 24, line 420: are “Sicels” the same as “Sikels”? Check if you mention either before this page ” to clarify)p. 25, line 443: change “offer” to past tense (offered) (Author response: DONE)p. 26, line 462: add space after period, before “Foreign” (Author response: DONE)p. 26, line 468: change semicolon to comma after “Stamer” (Author response: DONE)p. 28, ref. 1: remove the “Available from: ...” (Author response: DONE)p. 29, line 550: capitalize second initial for Ezzo, uncapitalize “Of” in journal name (Author response: DONE)p. 29, line 564: remove space before colon (Author response: DONE)p. 30, line 572: remove the “Available from: ...” (Author response: DONE)p. 30, line 585: remove the “Available from: ...” (Author response: DONE)p. 30, lines 609-10: capitalization of article title inconsistent with others, check the style expected. (Author response: DONE)p. 31, line 633: remove space after hyphen so that it reads “Look-Up” (Author response: DONE)p. 31, lines 635-7: remove the “Available from: ...” (Author response: DONE)Figure 3: the precision of the O isotope ranges should be consistent with decimal places, while given the precision of the measurements no more than 2 decimal places. Also the lower case “v” (Author response: DONE)Author response to journal requirements:1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. (Author response: DONE)2. We note that Figures 1-4 in your submission contain map/satellite images which may be copyrighted. Author response:https://commons.wikimedia.org/wiki/File:Mediterranean_Sea_16.61811E_38.99124N.jpg), taken from NASA’s globe software World Wind. The plan of Himera was replaced with a new image of Himera created by author SV. The base map of sites in Sicily was created in QGIS by author KLR.Figure 1 was edited to remove copyrighted images. The original Mediterranean Sea image (from Google Maps) was replaced with a public domain image from the Wikimedia Commons :Reviewers' comments: 18 Mar 2021PONE-D-20-37019R1 Isotopic evidence for geographic heterogeneity in Ancient Greek military forces Dear Dr. Reinberger:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Mario Novak Academic EditorPLOS ONE


I2 = 15,19%, Cochrane Q = 3,54 with overall estimate significance of p < 0,001. Rheumatoid arthritis is associated with over twofold risk of developing HRVA, and therefore, vertebral arteries should be meticulously examined preoperatively before performing craniocervical fusion in every RA patient.Rheumatoid arthritis (RA) might lead to atlantoaxial instability requiring transpedicular or transarticular fusion. High-riding vertebral artery (HRVA) puts patients at risk of injuring the vessel. RA is hypothesized to increase a risk of HRVA. However, to date, no relative risk (RR) has been calculated in order to quantitatively determine a true impact of RA as its risk factor. To the best of our knowledge, this is the first attempt to do so. All major databases were scanned for cohort studies combining words “rheumatoid arthritis” and “high-riding vertebral artery” or synonyms. RA patients were qualified into the exposed group (group A), whereas non-RA subjects into the unexposed group (group B). Risk of bias was explored by means of Newcastle-Ottawa Scale. MOOSE checklist was followed to ensure correct structure. Fixed-effects model (inverse variance) was employed. Four studies with a total of 308 subjects were included in meta-analysis. One hundred twenty-five subjects were in group A; 183 subjects were in group B. Mean age in group A was 62,1 years, whereas in group B 59,9 years. The highest risk of bias regarded “comparability” domain, whereas the lowest pertained to “selection” domain. The mean relative risk of HRVA in group A (RA) as compared with group B (non-RA) was as follows: RR = 2,11 , The online version of this article (10.1007/s10143-020-01425-w) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting synovial joints as well as other organs with high morbidity and increased mortality . Among mTwo researchers  scanned the following databases: PubMed MEDLINE, Web of Science, EMBASE, SciELO, and China National Knowledge Infrastructure. Search terms included “rheumatoid arthritis” AND “high-riding vertebral artery,” “high-riding vertebral groove,” “axial isthmus,” “high-riding transverse foramen,” “C2 isthmus,” “high-ride vertebral artery,” “HRVA,” “axis isthmus,” and “vertebral artery.” Time span was not restricted. The authors strictly followed MOOSE checklist . Titles were imported into Mendeley Desktop 1.19.4, which was also used for creation of the bibliographic data. Next, de-duplication was conducted. References of the included articles were tracked in order to identify any potentially missed papers.The following inclusion criteria were imposed: (1) cohort studies with exposed and unexposed groups, (2) patients with rheumatoid arthritis as an exposed group, (3) subjects without rheumatoid arthritis as an unexposed group, (4) information on incidence of HRVA per person, and (5) Neo’s and Bloch’s definition of HRVA . Of note, the above-mentioned measurements incorporated into the definition of the HRVA had to be taken in sagittal section of computed tomography (either classic or CTA). Exclusion criteria were determined as (1) groups who were operated on by means of unilateral C1-C2 transarticular fixation , (2) papers with overlapping data, (3) case reports, (4) cohort studies with a total number of patients less than 10, (5) letters to the editors, (6) commentaries, (7) conference abstracts, (8) insufficient data, (9) no definition of HRVA, (10) reviews, (11) surveys, and (12) errata. English language was not a condition—articles in other languages were translated by proficient speakers of the given language and then evaluated by the authors. In order to avoid data overlap, studies of the same authors were given a particular attention so as to identify any overlapping recruitment timeline or inclusion criteria.Two authors did the data extraction independently into the spreadsheet of Microsoft Excel 2016 . Pieces of information aimed to be extracted were as follows: (1) number of subjects without RA, (2) number of subjects with RA, (3) number of non-RA subjects with HRVA, (4) number of RA patients with HRVA, (5) geographical location, (6) age, and (7) sex. If an article was lacking important data, corresponding authors were communicated with.Quality check and risk of bias assessment of the studies included in this meta-analysis were conducted by means of Newcastle-Ottawa Scale (NOS) as well as by evaluation of the funnel plot symmetry. Two researchers  assessed each of the included studies separately. In case of discrepancy, a senior neurosurgeon (LS) was called in so as to reach consensus. All articles were evaluated based on three domains: (1) selection, (2) comparability, and (3) outcome. A maximum of stars that could have been awarded for each domain was as follows: for “selection” four stars, for “comparability” two stars, and for “outcome” three stars. Lack of any stars in a given domain meant the risk of bias was high. At least one star but shy of a maximum meant the risk of bias of moderate. A maximum number of stars meant the risk of bias was low. For the sake of “outcome” domain in NOS, an adequate period of the established diagnosis of RA was arbitrarily set to at least 2 years.I2 and chi2. I2 value was interpreted with general accordance: 0 to 40%, not important; 30 to 60%, moderate heterogeneity; 50 to 90%, substantial heterogeneity; and 75 to 100%, considerable heterogeneity. Level of significance for Cochrane Q p value was arbitrarily set to < 10% . Level of significance for p value of comparative tests was arbitrarily set to < 5% . For heterogeneity lower than 40%, fixed-effects model was planned. In case of heterogeneity ≥ 40%, random-effects model would be adapted.Meta-analysis was conducted by means of MetaXL 5.3, EpiGear International Pty Ltd.  and Statistica 13.3.0, TIBCO Software Inc. . Relative risk  was calculated with corresponding confidence interval of 95%. Heterogeneity was assessed based on A flow diagram shows steps of the selection process Fig. . Of 346 Summary of the Newcastle-Ottawa Scale for each study included in the meta-analysis is shown in Table I2 = 15,19%, Cochrane Q = 3,54, p = 0,32. Test for overall effect was significant with p < 0,001. The forest plot indicates mean distribution of RR across the studies along with the pooled effect , 7–3,05, IAlthough several papers indicated that RA might pose a risk of developing HRVA, some small studies stated to the contrary. In a small study of Lee J et al., none of three RA patients had HRVA . Their sFindings of this meta-analysis might be elucidated by progressive reduction of volumetric bone mineral density . RA is kRheumatoid patients’ atlantoaxial joints are prone to inflammation, subluxation, and dislocation , 21. ForA small number of studies are a limitation of the present meta-analysis. One potentially contributory cohort study of Miyata et al.  had to bRheumatoid arthritis is associated with an increased risk  of developing a high-riding vertebral artery. Therefore, VAs should be thoroughly examined on CT angiograms before performing craniocervical fusion in these patients. The presence of HRVA might influence choice of approach. More research studies with better methodology encompassing subjects outside Asia are needed in order to extrapolate the results.ESM 1(DOC 71 kb)