Listeria spp. and L. monocytogenes in four plants producing PDO Taleggio cheese. A total of 360 environmental samples were collected from different areas during production. The sampling points were identified as Food Contact Surfaces (FCS), transfer-Non Food Contact Surfaces (tr-NFCS), and non-transfer-NFCS (non-tr-NFCS). Fifty-nine ingredients/products were also analyzed. Listeria spp. was found in all the plants with a mean prevalence of 23.1%; plants that included a ripening area showed significantly higher prevalence if compared to the other plants. The positivity rate detected on FCS was moderate (~12%), but significantly lower if compared to NFCS . Among the FCS, higher prevalence was revealed on ripening equipment. Listeria spp. was never detected in the ingredients or products. A total of 125 Listeria spp. isolates were identified, mostly as L. innocua . L. monocytogenes was detected only from two FCS samples, in an area dedicated to the cutting of ripened blue cheeses; strain characterization by whole genome sequencing (WGS) evidenced a low virulence of the isolates. The results of the present study stress the importance of Listeria spp. management in the dairy plants producing PDO Taleggio and similar cheeses, mainly by the application of strict hygienic practices.The present study evaluated the presence of  Listeria monocytogenes remains a food safety challenge for cheese production plants: dairy products contaminated by this pathogen have been associated to some strong-evidence listeriosis outbreaks that occurred in Europe in the last decade  sta073/2005 ; in addi073/2005 . In 2017L. monocytogenes may survive and persist in dairy processing environments, or even grow when they find a favorable substrate, as in several dairy products [Thanks to its ability to replicate at refrigeration conditions and tolerate high salt concentrations, low water activity below 0.92), and a wide range of pH values (4.0–9.5), it is not surprising that products ,9.2, and a L. monocytogenes may also occur at several stages of dairy production and can be due to the use of contaminated ingredients or products (such as starter cultures or brines), but it is mainly caused by the spread from contaminated environment, utensils, and equipment such as floors, drains, shelves, clothes, brushes, coolers, etc. [Dairy products contamination can be considered a multifactorial event. Many sources are involved: raw milk can be contaminated at farm level and lead the contamination in the cheese-processing environment ; this evrs, etc. ,12. Prevrs, etc. ,14,15.L. monocytogenes in cheese production plants during processing but also after cleaning and disinfection procedures [L. monocytogenes strains up to 10 years in the food industries [Focusing on the production environment, several studies have already shown the presence of ocedures ,17,18,19ocedures , thanks ocedures . Many stdustries ,12.L. monocytogenes should not exceed 100 CFU/g during the shelf-life of RTE foods such as dairy products. In addition, L. monocytogenes should not be detectable in the product samples (n = 5.25 g each), if the foodstuff is able to support its growth, when it is still under the control of the producer, who cannot also demonstrate, to the satisfaction of the competent authority, that the counts will not exceed the limit of 100 CFU/g throughout the shelf-life. In order to apply an efficient control of the pathogen, processing areas and equipment should be monitored in addition to cheese testing; as other Listeria species (in particular L. innocua) share the same ecological niches with L. monocytogenes, it is recommended to enlarge the research in dairy plants to Listeria spp. to investigate on the presence of ecological niches suitable for the survival of the pathogen [According to EU Regulation 2073/2005 , the numpathogen .Listeria spp. and L. monocytogenes within the processing environment of four different plants involved in the production chain of PDO Taleggio cheese: three producers and one ripening plant. Taleggio is a PDO cheese produced in a limited area of Northern Italy, but is marketed worldwide. For the production, calf rennet and bacterial starters (Lactobacillus bulgaricus and Streptococcus thermophilus) are added to whole cow milk (raw or mainly pasteurized). The curd obtained is distributed into molds and submitted to resting  and to dry- or brine-salting. Then, the cheese is ripened for a minimum of 35 days on wood axes or boxes at refrigeration temperatures; during this time, it is weekly turned and sponged with brine [Listeria monocytogenes. This contamination could be of concern, especially considering that Taleggio rind is intended for consumption; thus, tracing the contamination within the plants could contribute to the hygienic management of the production process.The aim of the present study was to detect the presence of Samples were collected from four different dairy plants during three sampling sessions occurred in May, July, and October 2017. Briefly, dairy plant “A” produces PDO Taleggio, other PDO cheeses , and non-PDO blue cheese; it is intended for both production and ripening. Dairy plant “B” produces various PDO cheeses , other cheeses that need to be further ripened, and ricotta: ripening is done in plant “D.” Dairy plant “C” only produces PDO Taleggio cheese and a small amount of whey-butter; ripening occurs in plant “D.” Plant D only has premises for ripening and packaging different cheeses, such as PDO Taleggio, PDO Gorgonzola, PDO Strachitunt, and others, supplied by several producers (not only “B” and “D”).Environmental samples were collected during the dairy production process: a total of 360 environmental samples were collected from different areas . The sampling points were identified as Food Contact Surfaces (FCS), transfer-Non Food Contact Surfaces , and non-transfer-NFCS (non-tr-NFCS) . In particular, considering the production areas, sampling was performed on tables, drains, instruments, boxes, filters, manhole covers, washing machines, floors, scales, and operators’ boots. Samplings from the ripening areas involved boxes, brushes, packaging machines, sheets, and operators’ boots. Moreover, when present, other rooms/areas not directly involved in Taleggio production were sampled . In addition to environmental samples, a total of 59 ingredients , curd (10) and product samples  were taken.Listeria spp. and L. monocytogenes, sterile sponges pre-wetted with 10 mL of Buffer Peptone Water (BPW)  were used to swab surfaces, when possible, of about 10 × 10 cm2. After sampling, the sponges were immediately put into the original sterile bag and kept refrigerated. For the enumeration of Listeria spp. and L. monocytogenes, the same points (near the ones used for the detection) were sampled with pre-wet sponges following the same procedure. All the samples were immediately transported in refrigeration at the laboratory and analyzed within 24 h of collection.For the detection of Listeria spp. and L. monocytogenes, respectively. For enumeration purposes, aliquots of 90 mL of BPW were added; appropriate 10-fold dilutions were prepared and spread onto Palcam Agar (for Listeria spp.) and on ALOA (for L. monocytogenes). From each positive sample, at least one to three presumptive Listeria spp. and L. monocytogenes colonies were taken and streaked onto Triptic Soy Agar  and incubated at 37 °C for 24 h. Each colony was confirmed after gram staining, catalase and oxidase test, hemolytic activity and biochemical test Microgen Listeria-ID (MID67) .For bacterial detection samples were analyzed according to ISO 11290-1:2017 . After iL. monocytogenes by biochemical methods were subjected to DNA extraction, molecular serogrouping and WGS to define in silico Clonal Complex (CC), Sequence Type (ST), cgMLST, and relevant genes absence/presence, as previously described by Torresi et al. [TM, Thermo Fisher Scientific, Waltham, MA, USA). Then, 100 µL of 20 mg/mL lysozyme was added and incubated for 2 h at 56 °C. Finally, 300 µL of the suspension were transferred to the cartridges provided by the Maxwell 16 Cell DNA Purification Kit . DNA extraction was accomplished following the manufacturer’s instructions. DNA was quantified using a Qubit dsDNA HS (High Sensitivity) Assay Kit  and purity was checked by a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).Strains identified as i et al. . BrieflyTM Safe DNA Gel Stain (Thermo Fisher Scientific).Molecular serogroup-was performed using a multiplex PCR assay according to Doumith et al. and Kerouanton et al. ,26. BrieWGS was performed to define in silico Clonal Complex (CC), Sequence Type (ST), cgMLST and relevant genes absence/presence.L. monocytogenes. Then, genomes were uploaded into the Pasteur Institute platform (https://bigsdb.pasteur.fr) [Strains DNA were sequenced using the NextSeq500 Illumina platform using the Nextera XT protocol. Quality control was performed by FastQC  and trimteur.fr)  in orderp < 0.05.Data were submitted to chi-square test or exact Fisher’s test using PRISM graph pad 6 ; the prevalence data obtained from the different plants, during the three sampling sessions, and the different areas within each plant were compared. A comparison was also made among FCS, tr-NFCS and non-tr-NFCS samples. The threshold for statistically significant differences was settled at Listeria spp. and L. monocytogenes in the samples are summarized in Listeria spp. was found in all the plants in at least one sampling session , with a mean prevalence of 23.1% . This result was strongly influenced by the presence, in plants A and D, of the cheese ripening area. Listeria monocytogenes was found in only two samples from plant D (6.9%).The results obtained from the detection of of 23.1% , thus inWhen considering the same areas in the different dairy plants, no significant differences were shown related to the production (plants A-B-C) or ripening (plants A–D) areas: these results confirm the important influence of the production or ripening phase rather than the producer considered, taking into account that all the plants apply currently good manufacturing and sanitation practices. Focusing on the ripening phase, the entry of cheeses from different plants did not represent a particular concern, as the prevalence in plant D, which receives unripened Taleggio from various plants , was lower (if not significantly) than that observed in plant A, whose ripening area was dedicated exclusively to the cheese produced in the same plant.Listeria spp. prevalence (p < 0.05) in the plants including a ripening area (A and D), whereas this influence was not evidenced in the plants dedicated only to the production phase (B and C). When considering the areas separately, a significant difference was evidenced only in the samples from the ripening one, with data obtained from samplings performed in October showing higher counts than those taken in July and in May (p < 0.05). Thus, this finding could not be related to a different initial contamination rate of the product; as ripening environmental conditions are strictly maintained, the possible role of management procedures during this phase should be further investigated.The sampling session showed a significant effect on evalence . In partListeria spp. was detected in more than half of the samples) if compared to pasteurization, brining, packaging, and production areas. This could be explained by the high risk of contamination of the product in the ripening rooms from the environment, which could also be increased by cross-contaminations when commingled products from other plants are introduced. The ripening rooms are very often used for the aging of various typologies of cheeses that share the same needs for environmental parameters; thus, the cross contamination among different products is not unlikely. The environmental conditions of the ripening rooms, such as low temperatures and high ambient humidity, coupled with the surface type, and their contact time with the product may enhance the possibility for L. monocytogenes to persist and be selected. In this area frequent handling of the product  also occurred, which can contribute to the transfer of pathogens, with equipment or devices (brushes) that are difficult to sanitize and where the structures cannot be submitted to daily sanitation .Considering the different areas dedicated to the phases of PDO Taleggio production within each plant , the datAs expected, lower positivity rates were revealed in the production and in the milk pasteurization areas, thanks to the absence of ripened product and to the efficacy of the daily cleaning and disinfection procedures, thus limiting the presence of suitable ecological niches.p < 0.01), as expected. Considering the different areas of the plants, the lower rate on FCS was mainly evidenced in the ripening areas (p < 0.05); the comparison among the three sampling sessions showed that the higher prevalence observed during the last session , transfer-Non Food Contact Surfaces (tr-NFCS), and non-transfer-NFCS (non-tr-NFCS). As shown in L. monocytogenes showing 80% of positivity; in particular, they selected 16 strains and traced a contamination of the salting equipment. A particularly high prevalence of Listeria spp. was detected in equipment used for Taleggio rind brushing (6/8 positive samples on brushes and brushing tables). These data are in agreement with those obtained with other dairy products submitted to surface washing (positivity rate of 44–72%) [Listeria spp. was detected in any of the areas of the plants (0/14 samples), thus indicating the compliance with the personnel hygiene recommendations.Among the FCS, a very low positivity rate was recorded on equipment/surfaces located in the production and brining areas, whereas a high prevalence was detected on equipment used for ripening like brushes, tables, boxes, boards, and cloths (8/26 positive samples) . In a pr 44–72%) ,18,19, hListeria spp. was detected for some typologies , with only 3/37 positive samples  and especially from the drains (36/109); in applying the procedures of the HACCP plan, the choice of the drains as monitoring points should be regarded as an effective method to reveal the presence of Listeria within a dairy plant. With the same aim, the sampling of the overshoes used during the sampling activity, reflecting the situation of the floors of all the production, ripening, and packaging areas, demonstrated to be an even more sensitive method, resulting in a very high prevalence of Listeria spp. (11/18 samples).Considering the results obtained from the non-tr-NFCS surfaces, a low prevalence of  samples . An inteA particular concern could be posed by tr-NFCS surfaces, as they can act as by-pass points for the contamination of different working areas: the highest prevalence among these samples was detected on trolley wheels (4 out of 16 samples), followed by operators’ boots (3/17) and doors/handles (1/7) . It has L. monocytogenes [The presence of various production processes into the same plant should be carefully considered when evaluating the contamination patterns: in this study, three areas dedicated to the production of different dairy products were considered: the contamination of ricotta and butter production rooms  showed a very low contamination rate . This result was expected, as both butter and ricotta production processes are characterized by a low contamination probability if considered alone, but in particular ricotta must be protected by the post process contamination coming from other working areas, as it represents a suitable growth substrate for ytogenes .Listeria spp. contamination rates, both from FCS (3/9) and drains (3/5)  and other products obtained in the same plant (due to the possible transfer of the microorganism). In this study, despite the proximity to the Taleggio packaging area, a spread of L. monocytogenes from this section was not observed, suggesting the importance of good production practices in segregating the contamination within complex plants.The room dedicated to the piercing, cutting, and packaging of blue cheeses (mainly PDO Gorgonzola) showed relatively high ns (3/5) ; in addiListeria spp. was never detected in the ingredients  or in the product, both considering the curd and Taleggio; these results confirm the importance of the application of proper procedures to manage the presence of these microorganisms in the production plants (as it cannot be completely avoided in the current process conditions) and to prevent the risk for consumers.Finally, Listeria spp. counts were below the enumeration limit of the method (1 CFU/cm2), except for some sampling points , where counts between 1 and 1000 CFU/cm2 were recorded. These samples were obtained only from plants A and D , confirming the difference already noticed in the bacterial prevalence. High counts were mainly obtained from the ripening areas, characterized by environmental conditions supporting the selection and slow growth of Listeria spp., and on floor drains, where microorganisms are easily collected during the cleaning procedures. In drains, counts varied from 1 (drains in packaging) to 700 CFU/cm2 (drains in blue cheese piercing machine). Three samples with high Listeria spp. counts were taken from FCS, and in particular from brushes (20 and 240 CFU/cm2) and a table where cheese blocks were laid during brushing operations (86 CFU/cm2), confirming the need for particular care during this periodical production phase of PDO Taleggio. In blue cheese, piercing machine values of 110 CFU/cm2 were enumerated, while in sampler overshoes, a value of 1 CFU/cm2 was enumerated.In most of the positive samples, Listeria spp. isolates were submitted to species identification , L. grayi, L. welshimeri, L. seeligeri, and L. monocytogenes. The diffusion of the different Listeria species among the sampling points was also evaluated. As shown in L. innocua was predominant in all the areas of the plants (63 out of 83 positive samples hosted L. innocua); apart from the abovementioned presence of L. ivanovii, the two most contaminated areas showed the presence of a diversified Listeria spp. population, namely the ripening area and the one dedicated to piercing, cutting, and packaging of blue cheeses. These data confirm the importance of cheese ripening for the selection and persistence of Listeria, thanks to the environmental conditions and to the possibility to survive steadily on the surface of the equipment and potentially on the cheese blocks/wheels.A total of 125 fication : they weListeria spp. may act as a sentinel for the presence of the pathogenic strains as they share the same ecological niches. Moreover, although Listeria innocua was initially considered non-pathogenic, its role has been debated, as it was isolated at least once from a fatal human case [In any case, as stated previously, the presence of man case .L. monocytogenes strains accounted for 1.6% (n = 2) of the overall isolates (n = 125). Both strains were isolated from the blue cheese production area and were characterized as belonging to serogroup IIa. Molecular characterization “in silico” revealed genetic proximity of the two isolates. Both strains were classified as CC31, ST325 and showed the same virulence, antibiotic resistance, and stress resistance genes. In particular, among 92 loci classified in the Pasteur Institute platform as belonging to virulence, 51 were detected, comprising a complete Listeria Pathogenicity Island 1 (LIPI 1) and InlB, E, F, H, J, K. InlA was detected but showed the same single point mutation in both strains. As regard the stress islands, four of the five genes belonging to SSI 1 (lmo0444-lmo04448) were detected as exact match while gaps or two single mutations were recorded for the gene lmo0445. No metal and detergent resistance genes were detected. Core genome MLST analysis showed three allele differences due to the lack of three loci in one of the isolates. Characterization results highlighted the finding of hypovirulent strains, a 2017 EFSA opinion [L. monocytogenes strains were rarely found in humans and are usually present in meat and meat products. CC31 strains were, furthermore, found among non-hemolytic strains [As shown in  opinion ,36 showe strains . As specListeria spp. in different PDO Taleggio producing plants, with very low prevalence of L. monocytogenes. The data analysis showed the importance of the ripening areas, of which the role was quite independent from other factors, such as the plant or the origin of the curd. A moderate prevalence of Listeria spp. on the Food Contact Surfaces: in this light, it has to be considered that the contamination of the cheese mass by L. monocytogenes is unlikely; however, the rind contamination  can in any case be considered of concern, as PDO Taleggio rind is defined to be edible, and a link between this cheese and listeriosis outbreaks has been hypothesized in some cases. The isolation of L. monocytogenes strains from the plant environment, even if hypo-virulent, confirms the importance of the application of control procedures along the production and ripening process. Moreover, the identification of clones previously isolated from a totally different food production chain puts the focus on the ability of this pathogen to adapt to various hurdles, thus colonizing different environments.The results of the present study show the diffusion of L. monocytogenes in a dairy processing plant is crucial for the application of corrective and preventive measures and for the traceability of outbreak clones. Moreover, the overlay of the production areas dedicated to different products is likely present in several dairy plants, making possible the cross contamination of the final products. Three of the plants considered in this study produced several different cheeses other than PDO Taleggio; such products are potentially affected by the same risk factors for the growth of Listeria monocytogenes . In some cases, such products can act as additional niches for Listeria persistence within the plants or as vehicles for the contamination of PDO Taleggio itself. In this light, particular attention must be posed to some transfer surfaces, which can favor the circulation of the microorganism among areas dedicated to different dairy products and production phases. Thus, a careful cleaning and disinfection program, as well as the proper management of personnel, equipment, and product flows, must be applied. A constant microbiological monitoring program in the plant should be also addressed as one of the main control points in a dairy plant. In such program, Listeria spp. can act as a sensitive sentinel for the identification of potential survival niches, that could be hardly identified by the only research of L. monocytogenes.The identification of the potential sources of Listeria spp. management in the dairy plants producing PDO Taleggio and similar cheeses, mainly by the application of strict hygienic practices (involving both sanitation and production procedures), the implementation of operator training, and the management of personnel, equipment, and product flows, in order to guarantee product safety.The results of the present study, performed in medium scale plants that currently follow specific hygiene procedures, stress the importance of


Moreover, the VD_fApEn (90%) reached a higher OSA screening accuracy compared with LF/HF ratio (80%) and fApEn (78.3%). Therefore, VD_fApEn provides a potential clinical method for ANS fluctuation analysis in OSA patients and OSA severity analysis.Obstructive sleep apnea (OSA) is a fatal respiratory disease occurring in sleep. OSA can induce declined heart rate variability (HRV) and was reported to have autonomic nerve system (ANS) dysfunction. Variance delay fuzzy approximate entropy (VD_fApEn) was proposed as a nonlinear index to study the fluctuation change of ANS in OSA patients. Sixty electrocardiogram (ECG) recordings of the PhysioNet database  were intercepted for 6 h and divided into 5-min segments. HRV analysis were adopted in traditional frequency domain, and nonlinear HRV indices were also calculated. Among these indices, VD_fApEn could significantly differentiate among the three groups ( Obstructive sleep apnea (OSA) has been widely reported as a potentially fatal respiratory disease that occurs in sleep. It is characterized by recurrent disorder of upper respiratory tract, with clinical manifestations of daytime sleepiness, snoring, and decreased sleep quality . OSA wasHeart rate variability (HRV) is a kind of noninvasive method which can effectively evaluate ANS function in OSA patients ,6,7. Thep < 0.05) [p < 0.05) [p < 0.001), and LF/HF was regarded as the most useful index to evaluate the AHI degree of OSA patients [In frequency domain analysis, low frequency power (LF) and high frequency power (HF) index can indicate the balance controlling of ANS. Song et al. found that the LF/HF index displayed the best statistical significance in HRV indices ( < 0.05) . Urbanik < 0.05) . Park etpatients . It can p < 0.05) [Entropy methods can be used to estimate the complexity of HRV. In general, normal systems have inherent complexity, while the complexity of system in most patients has downward trends ,19,20. H < 0.05) . As a me < 0.05) . These fHowever, the changes of ANS in OSA patients are also affected by daily activities and individual differences . The shoTherefore, the variance delay fuzzy approximate entropy (VD_fApEn) was proposed in this paper. It is a nonlinear HRV analysis method which can be used for further dynamic analysis of the ANS in OSA patients. First, the small-scale variance of successive RR sequences was firstly extracted to reflect the tension of ANS. Then, by applying fuzzy entropy (fApEn) combined with delay method, VD_fApEn was calculated reflecting the disorders of short-term fluctuations in ANS. Finally, the VD_fApEn was used for OSA screening and analysis of OSA severity. Further details will be given later in this article.www.physionet.org, which provides the Apnea-ECG data on obstructive sleep apnea. This database contains 70 single-lead ECG signals with 100 Hz sampling frequency, and the lengths varying between 401 and 587 min. Each recording includes minute-by-minute annotations to indicate the presence or absence of apnea during that minute, which were made by human experts.The database used in this paper for OSA analysis was downloaded from According to the number of minutes with apnea, the recordings in the PhysioNet database were classified into three categories: A, B, and C. Recordings having fewer than 5 min of disordered breathing were defined as class C . Recordings containing at least 1 h with an apnea index of 10 or more and at least 100 min of apnea were classified as class A . Between these two groups, recordings containing at least 1 h with an apnea index of 5 or more and between 5 and 99 min with apnea during the recording were defined as class B .According to these criteria, 70 recordings of 32 subjects were classified as follows: 40 class A recordings, 10 class B recordings, and 20 class C recordings. In this study, 10 borderline recordings of class B were excluded, and a total of 60 recordings from class A (20 recordings) and class C (40 recordings) were used. Apnea-hypopnea index (AHI) is an indicator of the severity of OSA, defined as the number of apneas and hypopnea events per hour during sleep. The 60 recordings were further divided as following groups according to AHI as follows: normal group , mild-moderate OSA group , and severe OSA group .First, the 60 full-night ECG recordings were intercepted in 6 h for the regularization of data. Then, RR segments (RRs) were extracted from the ECG signals with the algorithm proposed by Pan and Tompkins , and theLow frequency power , high frequency power , and the ratio of LF power and HF power (LF/HF) were computed as HRV frequency indices. These indices are obtained with the fast Fourier transform method to calculate the power spectral density of these RRs.Fuzzy Approximate Entropy (fApEn): The fApEn is an effective and robust algorithm for measuring the complexity of a discrete sequence, used for assessing the complexity of physiological signals . The det(a) Given an (b) The maximum absolute distance (c) Define the similarity degree (d) Then, calculate the average similarity from each vector to another as follows:(e) Construct (f) Finally, the fApEn is defined as:For a finite discrete series, fApEn can be calculated as:Varience Delay Fuzzy Approximate Entropy (VD_fApEn): First, the variance of RR sequence in small scale was calculated to reflect the fluctuation change of HRV. As shown in (a) Given an (b) Coarse-grain (c) To embed the delay method into the fApEn method, we constructed The following steps are the same as the fApEn calculation steps (2)–(7).N and r for entropy indices of the HRV signal. Therefore, the performances of fApEn and VD_fApEn with changes of parameters were compared using simulated signals in this study. Three kinds of nonlinear signals with different complexity are generated as MIX , which are defined as follows:Considering that the ANS is highly nonlinear and complex, a nonlinear signal system model was constructed to verify the feasibility of VD_fApEn for ANS analysis. In addition, it is necessary to choose appropriate m, N, and r, should be focused. m is the dimension of vector sequences and was proved to be suitable set as 2 [N and r settings in the complexity measure, we compared the variation of fApEn and VD_fApEn with N and r. For the selection of parameter N, r, r ranged from 0.1 to 1.In the calculation of entropy, three parameters, set as 2 ,30. To fp < 0.05 represents statistically significant, and the statistical results of indices are showed as mean ± SD. For OSA screening, Fisher’s discriminant function of SPSS was used, and the overall accuracy, sensitivity, and specificity of HRV indices were presented [HRV indices were calculated using MATLAB . All statistical analyses were adopted using SPSS . The HRV indices data calculated in this study were analyzed in SPSS, and results showed that the data conforms to normal distribution. HRV indices were evaluated by one-way ANOVA followed by post hoc analysis with the least significant difference (LSD) test for analysis among the control group, mild-moderate OSA group, and severe OSA group. resented .Monotonicity, consistency, and continuity are important references used in complexity analysis to reflect the performance of entropy indices. Monotonicity refers to the monotonic tendency of parameter variation; consistency means that if a sequence is more complex, all values of entropy for testing should be larger; continuity means that there should be no mutation when the parameters change slightly .n value is greater than 200, both fApEn and VD_fApEn reached relatively stable. VD_fApEn made a better distinction among Mix  signals. As shown in The results of frequency domain and nonlinear indices for the normal, mild-moderate OSA and severe OSA group are shown in As shown in In calculation of the VD_fApEn, variance of RR sequence in small scale was calculated to reflect the fluctuation change of HRV. In this study, the results of VD_fApEn applied to OSA disease severities analysis, and the results of OSA disease screening were analyzed, as RR scale ranged from 2 to 9.In This paper verified the performance of HRV frequency domain indices by comparing with previous studies. LF/HF can reflect the status of autonomic nerve control and is regarded as a reliable indicator providing valuable information in apnea events discrimination . Resultsp < 0.001), while the index LF/HF and AHI were positively correlated . It can be observed that there is a better correlation between VD_fApEn and AHI. In addition, HRV indices have been used for OSA screening in previous studies. Haitham et al. reported that sample entropy of OSA patients had less complex HRV pattern, and the accuracy of sample entropy for OSA screening reached 70.3% [In this study, VD_fApEn could significantly differentiate between any two groups among normal, mild-moderate OSA and severe OSA group compared with LF/HF and fApEn . We alsoed 70.3% . The slied 70.3% . In thisHRV analysis is widely adopted in ANS function evaluation, but the instantaneous change of ANS was ignored. Considering that HRV is the result of many nonlinearly interacting processes, nonlinear HRV methods are suitable for HRV analysis. As shown in In this study, the small-scale variances of successive RR sequences were extracted to reflect the instantaneous tension of ANS, and fApEn combined with delay method was adopted to reflect the complexity of ANS tension. Studies have shown that the strong correlation between continuous heartbeat may mask the non-linear measurement, and the occurrence of apnea events may have a delayed effect on ANS . Thus, dParameters of VD_fApEn were also analyzed in this study. The scale of RR sequences for calculating variance was analyzed, and it could be inferred from the results that the ability of VD_fApEn to distinguish OSA disease severity increased with scale decreased. When the scale reduced to 5, the OSA screening accuracy of VD_fApEn reached its maximum (90%) . TherefoOSA is a common respiratory cardiovascular syndrome during sleep. It has been extensively studied that abnormality of ANS existed in OSA patients, and HRV analysis was reported to be an effective method to evaluate the function of ANS . ConsideResearch showed that the fluctuation of respiratory and cardiovascular may lead to disorder of HRV, and OSA was related to the activity and short-term state changes of the ANS . It can Present study still has some limitations for further improvement. First, personal factors of subjects may have impact on the results . Second, our algorithm should be improved in many ways, the proposed index also needs further verification. Finally, the existence of underlying cardiovascular disease and sleep related factors may have a certain impact on the HRV analysis of OSA patients, which was not considered. In future study, we will adopt a larger amount of data and further verify the reliability of the index proposed in this paper by combining polysomnography (PSG) indicators.In this study, variance delay fuzzy approximate entropy (VD_fApEn) was proposed as a nonlinear index to study the fluctuation change of ANS in OSA patients. When used for disease severity analysis of OSA, results showed that VD_fApEn could significantly differentiate between any two groups among the normal, mild-moderate OSA, and severe OSA group. The complexity of autonomic fluctuations decreased significantly with increased severity of OSA. When used for OSA screening, VD_fApEn had a higher accuracy compared with the traditional HRV indices. Therefore, VD_fApEn provides a potential clinical method for analysis of ANS alterations in OSA patients and severity of OSA disease.


Objective: We investigated the effects of lactoferrin (LF)-fortified formula on acute gastrointestinal and respiratory symptoms in children.Design: Randomized, double-blind, placebo-controlled trial.Setting and subjects: Children aged 12–32 months in Japan.Intervention: Intake of placebo or LF (48 mg/day)-fortified formula for 13 weeks.Primary endpoint: Prevalence of acute gastrointestinal and respiratory symptom.Results: One hundred nine participants were randomized. Eight participants were lost to follow-up, withdrew consent, or were deemed inappropriate for the trial, with 101 participants receiving complete analyses .Outcomes: The prevalence of acute gastrointestinal symptoms was significantly less in the LF group (22/53 [41.5%]) than in the placebo group . The total number of days having acute respiratory symptoms was significantly lower in the LF group (9.0) than in the placebo group .Harms: The rate of adverse events was similar between the groups. No adverse drug reactions were found.Conclusions: LF intake decreased the prevalence of acute gastrointestinal symptoms in children aged 12–32 months. Despite remarkable advances in the improvement of child survival, the mortality rate of children in developing countries remains high. In 2015, 5.9 million children worldwide did not live to the age of 5 years, primarily due to pneumonia (12.8%) and diarrhea (8.6%)  recommends that mothers breastfeed their children for up to 2 years of age or beyond . The advLactoferrin (LF) is an 80 kDa iron-binding mammalian glycoprotein of the transferrin family. It is a component of exocrine secretions such as breast milk and saliva and is present in neutrophil granules . LF playBreastfeeding is the simplest and most cost-effective intervention for protecting children against diarrhea and all causes of mortality . HoweverThis was a randomized (1:1), double-blind, placebo-controlled, parallel-group, comparative trial conducted by the Department of Preventive Medicine and Public Health of Shinshu University School of Medicine in Matsumoto, Japan. It was performed in accordance with the current revision of the Declaration of Helsinki  and EthiThis trial was performed between November 27, 2017 and April 1, 2018. Eligible participants were apparently healthy children between the ages of 12 and 32 months who attended nursery schools in Matsumoto, Japan. Exclusion criteria were allergies to milk or soybeans, receiving breast milk, habitual consumption of LF, history of serious disorders of the liver, kidney, heart, lung, gastrointestinal tract, blood, endocrine system, or metabolic system, or judged to be inappropriate to participate in the trial by the principal investigator.The participants were randomly allocated into a placebo or LF (48 mg/day) group. The participants' parents were instructed to reconstitute one sachet containing either the placebo or LF-fortified formula with water or boiled water cooled to 50°C and provide their children with one sachet per day during the 13-week intervention period. One sachet contained powdered formula, which was a modified commercial growing-up formula for children aged 12–36 months manufactured by Morinaga Milk Industry, Co., Ltd., Japan. Both the placebo and the LF formulas were composed of the same nutrients except for the addition of LF (48 mg) in the LF formula. LF was replaced by dextrin in the placebo formula. Both test formulas were similar in appearance, texture, smell, and packaging. During the intervention period and 2-week post-intervention period, formula intake , acute respiratory or gastrointestinal symptoms, any diagnoses made by an examining physician, the names of any medications, and any other physical changes were recorded in diaries by the parents based on previous researches , 15.Diarrhea (once or more loose or liquid stool per day), vomiting, and/or fatigue associated with them were recorded as acute gastrointestinal symptoms. Nasal secretion/congestion and/or cough/sputum, and/or fever (≥38.0°C) and fatigue associated with them were recorded as acute respiratory symptoms. All participants began and completed the administration of the test formula and diary records on the same day. Well-trained public health workers contacted the parents every 4 weeks at nursery schools to verify the intake of the test formulas and diary records, and carefully collected further information regarding symptoms of any acute infections or other issues such as allergic symptoms. Symptoms were confirmed by the principal investigator based on diary described by the parents and information from the public health workers, and ultimately determined to be due to respiratory infections, gastroenteritis, or other issues. If respiratory and gastrointestinal symptoms were considered to be complicated, they were counted in both cases. Vomiting due to coughing was considered a respiratory symptom.The intake of test formula on each day was recorded as 100, 50, or 0% of one sachet in the diary. Based on these data, the average intake per day during the test period for each participant was calculated. Compliance was expressed as the median of the average intake per day in the placebo or LF group. At the end of the test, the remaining test formulas were collected, and the records were confirmed to be consistent. Parents were instructed in advance so that only the test participants could drink the test formulas.The primary endpoints were the prevalence of acute gastrointestinal or respiratory symptoms based on diary records described by parents in the interventional period. Prevalence rate was defined as the number of participants who experienced more than a symptom. The secondary endpoints were the total number of days and duration of the symptoms during the interventional period. The total number of days was defined as the cumulative number of symptomatic days and duration was the mean of consecutive days per episode. Individual episodes were separated by at least 1 day.Any unfavorable or unintended signs, symptoms, or diseases were defined as adverse events. Adverse events were evaluated using the Revised National Cancer Institute - Common Toxicity Criteria Version 4.0, and those events where a causal relationship with the intake of the test formula were identified were designated as adverse drug reactions.Children attending nursery schools were reported to have a higher risk of gastrointestinal and respiratory infections than children cared for at home . HatakkaAn independent allocation manager at a different university produced a computer-generated allocation table using the permuted block method [block size of 4 (1:1 ratio)] according to nursery school. The allocation tables were sealed in an opaque envelope and were kept until key breaking. The investigators, subjects, and public health workers were blind to all information during this period. The investigators enrolled the subjects and assigned them to test formula numbers. Key breaking was performed after locking the database and statistical analysis plan.P-values <0.05 were considered statistically significant.Data are expressed as the mean ± standard deviation or the median (interquartile range). Fisher's exact test was used to analyze the prevalence of symptoms and the Mann-Whitney U test was employed to evaluate the total number of days and duration of any symptoms. Statistical analyses were performed using IBM SPSS Statistics version 24 software . n = 49; LF, n = 60)   . The mea n = 53) . There wp = 0.265). The numbers of participants who exhibited some symptoms were: total, 96 (95.0%); acute gastrointestinal symptoms, 52 (51.5%); and acute respiratory symptoms, 94 (93.1%).The median (interquartile range) intake rates of the test formula were comparable at 84.1% , and 75.3%  for the placebo and LF participants, respectively (p = 0.046), whereas the prevalence of acute respiratory symptoms was comparable between the groups during intervention period  (Escherichia coli O111 (verotoxin negative) in 1 child.Regarding the primary endpoints, the prevalence of acute gastrointestinal symptoms was significantly lower in the LF group (22/53 [41.5%]) than in the placebo group  . Fecal sp = 0.030), while the total number of days of gastrointestinal symptoms and the duration of acute respiratory and gastrointestinal symptoms were similar between the groups  than in the placebo group , while there was no significant difference in acute gastrointestinal symptoms  and 2 and 1, respectively, in the post-intervention period (p = 0.587). Major adverse events included eye discharge, asthma, and eczema. No adverse drug reactions were found.Thirteen participants in the placebo group and 11 in the LF group experienced adverse events during the intervention period (We herein described the first study to investigate the effects of an LF-fortified growing-up formula on acute gastrointestinal and respiratory symptoms in children aged 12–32 months. Our results indicated that an LF-fortified formula can reduce the prevalence of acute gastrointestinal symptoms and the total number of days of respiratory symptoms in children > 1 year of age.Gastroenteritis is mainly viral or bacterial. Worldwide, rotavirus is considered the most common viral cause of severe acute gastroenteritis, accounting for 453,000 deaths and over 2 million hospitalizations among children under the age of 5 years . AlthougLF exerts antiviral effects on rotavirus and norovirus through the inhibition of viral attachment and replication in animals , 23. Morin vitro (The common cold and influenza are the most common upper respiratory tract infections. In adults, consumption of LF-containing tablets reduced common cold-like symptoms , and LF in vitro , 30. In in vitro . No humain vitro .In this study, the prevalence and total number of days of acute respiratory symptoms were significantly lower in the LF group than in the placebo group, even during the post-intervention period. Ishikado et al.  reportedSome studies in adults have reported various beneficial effects of LF at 200 mg/day such as protective effect against infectious gastroenteritis , improveThis study had some limitations. First, the data regarding acute respiratory and gastrointestinal symptoms were collected from self-reporting diaries by the parents, which have resulted in a mingling of physician's diagnoses and subjective interpretations of symptoms as determined by the parents. However, in order to minimize the subjective bias, health workers periodically contacted parents and carefully collected information regarding each symptom, with the principal investigator comprehensively judging each symptom as an acute illness or something else , 15. SecIn spite of the above limitations, this is the first randomized, double-blind, placebo-controlled trial to investigate the effects of an LF-supplemented growing-up formula on acute respiratory and gastrointestinal symptoms in children aged 12–32 months. This investigation demonstrated that LF ingestion could decrease the prevalence of acute gastrointestinal symptoms and lower the total number of days of acute respiratory symptoms in children aged 12–32 months. It provides important evidence on the beneficial effects of LF for healthy child development.nmotoki@shinshu-u.ac.jp.The datasets analyzed in this article are not publicly available because participants of this study did not agree for individual data to be shared publicly. Requests to access the datasets should be directed to Noriko Motoki, The studies involving human participants were reviewed and approved by the Institutional Review Board of Shinshu university school of medicine. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.NM, KY, FA, and TN designed the study. NM, MMiz, TT, MMiy, and MT contributed to data collection. NM, SK, and HO performed statistical analysis and wrote the manuscript. TT, KY, FA, and TN provided critical feedback on the manuscript. All authors reviewed and approved the manuscript.MMiy, SK, HO, MT, KY, and FA were employed by Morinaga Milk Industry, Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, we use a recently developed Bootstrap ARDL model to examine the influence of longevity (life expectancy after giving birth) and alcohol consumption on economic progression (GDP) in both China and India during the years between 1992 and 2015. Empirical results have shown an extended link across economic development, longevity, and alcohol use in both China and India. The Granger causality test, derived from the Bootstrap ARDL model, demonstrates a unidirectional relationship between economic growth and longevity in China. However, a bidirectional causality exists between longevity and alcohol use in India. Results have important implications for Indian and Chinese governments' public health policies, focused on alcohol consumption reduction specifically, and population health generally. Many studies have recently explored the relationship between longevity (life-expectancy) and economic growth. However, few have focused on a causal link between longevity and economic growth. Causal linkage between longevity and economic growth is key for policymakers designing national health policies that promote a long-life. Considering the public health data, the question remains as to why people live longer than ever before? One possible reason, reported by previous studies, is that improved medical health leads to a long-life that supports economic growth. However, we can consider the causality runs in reverse—economic growth improves health. Against this backdrop, the current study revisits the impact of longevity on economic growth (GDP) in both China and India, between the years of 1992 and 2015, using a recently developed Bootstrap ARDL model.Over half of income inequality, between developed and developing countries, is associated with health and results in a short life expectancy . PhysicaEconomic growth is pro-cyclical for health progress, raising life expectancy and lowering mortality, as reported by Pritchett and Summers , Filmer Other studies, such as those by Dehejia et al.  and NeumTo better understand this complex relationship, we employ information from the World Bank, focusing on life expectancy in the two leading population nations of India and China. India's life expectancy has increased from 41.17 to 68.80 years, while China' has risen from 43.72 to 76.41 years. Both countries' GDP have increased from 47.21 and 42.16 billion USD, in 1962, to 12,237.70 and 2,650.73 billion USD, in 2017, for China and India, respectively. The difference in longevity between the two nations is now 7.61 years, and although GDP started at a comparable level, China's is now 4.62 times larger. Despite having World Class hospitals and medical practitioners, India experiences hygiene and medical education deficiencies when compared with China.According to the United Nations, China's population will expand until the year 2025. China's total birth and fertility rate has diminished swiftly since the 1970's, due to government policy, resulting in a changing age structure now over weighted to the elderly. In 2000, China became an aging society, with the proportion of the elderly population exceeding 7% for the first time . The highest increase in alcohol consumption, by 2025, is expected in the South-East Asia Region, with India increasing by 2.2 L and China by 0.9 liters per capita . UnderstThe bootstrap autoregressive-distributed lag (Bootstrap ARDL) test of the cointegration method by McNown et al.  was adopThe remainder of this paper is organized as follows: Section Theoretical Background and Literature Review describes the literature review and theoretical background. Section Data and Methodology presents the data used in this study, while Section Empirical Results and Policy Implications describes the Granger causality test results using a Bootstrap ARDL model along with policy implications of our findings. Section Conclusions concludes the paper.We can define our empirical model based on the neoclassic growth model of Zhang et al.  defined After some manipulation that we can write as the following log-linear form:Where y is economic growth rate, x is longevity (life-expectancy), and z is a control variable. We use alcohol consumption as a control variable as alcohol consumption affected health and economic growth in both countries, in turn impacting both longevity and economic growth.Previous studies demonstrate four causality paths between economic development and health. The income view is that increasing incomes will result in an improved overall population health. Theoretically, economic growth can provide governments with the necessary tools to build an improved health system and to invest in technologies, governance, education, and institutional quality. Many researchers, such as Pritchett and Summers , Filmer With the fixed effects Panel data model (from 1960 to 1985), focused on developing countries' child fatality rates, Pritchett and Summers  found thGerdtham and Johannesson  evaluateBaird et al.  found anTobacco and alcohol consumption increase with income levels . Moreovevia the entire male population of Sweden, aged between 20 and 64 from 1993 to 2007, showing a pro-cyclical relationship between the business cycle and fatality levels. Cutler et al.  test described by McNown et al. . This teIn general, a 2-variable ARDL based on Pesaran et al.  can be wF-test or t-test exists in cointegration.Equation (3) shows that Y to X are unrelated, illustrating that we did not permit more of the variables to be endogenous, causing it to subvert the preconceived distribution of the numbers illustrated by Pesaran et al. . The weaF-test and t-test of co-integration proposed by Pesaran et al.  to a 3-variable case defined as the follows:In the short run, the Granger-causality test for x y should capture the differences that lag on x along with the level of lag of x to identify if γ > 0 and δ = 0. For z y, there ought to exist the differences of lag on z as well as the level of lag of z to understand if ϕ > 0 and π = 0 (under cointegration situation).F-tests and t-tests for China and India in the GDP equation. For both countries, GDP serves as the dependent variable, while longevity and alcohol consumption are explanatory. Namely, longevity and alcohol consumption account for the commercial growth of both countries for the longest time. Whereas, the dependent degenerate is rejected for China and lagged, while the independent F-test and variable Fdep are unimportant, so the degenerate case #2 is not sufficient to set up the code in the long run. In other words, all three-test statistics, F-test, Findep, and Tdep must be significant, and we find this to be the case in the China and the GDP equation.(1) Results from Bootstrap ARDL Test–Cointegration TestResults of the Bootstrap ARDL cointegration tests show that longevity and alcohol consumption explain economic improvements in India and China in the coming few years. As (2) Granger Causality Test Results Based on Bootstrap ARDL Model and Policy ImplicationsFrom a long-term perspective, we found that a co-integration relationship exists between longevity and alcohol consumption within GDP equations for both China and India, suggesting that longevity and alcohol consumption affect economic growth. Developed economies and emerging economies exhibit a connection between GDP and longevity, with emerging countries showing a stronger relationship , 53. ManThe current study investigates the relationship across economic development, longevity, and alcohol use in India and China. Results show a unidirectional negative Granger causality related with both economic growth and longevity just as longevity and alcohol consumption are also negatively related in China. These results are consistent with Vaupel , Ruhm 1, 13, EcoThe datasets generated for this study are available on request to the corresponding author.FL: topic, data collection, introduction, literature review, results discussion, reference, and manuscript review. M-CW: methodology, results, manuscript review and correction. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The etiology of prostate cancer (PCa) remains largely unknown. Compliance with the 2018 World Cancer Research Fund/American Institute for Cancer Research (WCRC/AICR) cancer prevention recommendations and its relationship to PCa was evaluated. A total of 398 incident PCa cases and 302 controls were included. The selection criteria for both cases and controls were: (i) age between 40–80 years; and (ii) residence in the coverage area of the reference hospitals for 6 months or more prior to recruitment. A score to measure the compliance with the recommendations of 2018 WCRC/AICR criteria was built. The level of compliance was used as a continuous variable and categorized in terciles. The aggressiveness of PCa was determined according to the ISUP classification. Adjusted odds ratios (aOR) and their 95% confidence intervals (95% CI) were estimated using multivariable logistic regression models. A slight protective tendency was observed between the level of compliance with the preventive recommendations and PCa risk, aOR = 0.81 (95% CI 0.69–0.96) for the total cases of PCa. This association also was observed when the aggressiveness was considered. In addition, limiting consumption of “fast foods”, sugar-sweetened drinks, and alcohol were independently associated with lower risk of PCa. Prostate cancer (PCa) is the most common cancer in men, and has the highest incidence and the third highest mortality, after lung and colorectal cancer, in Europe . In receDespite its considerable impact, the etiology of PCa remains largely unknown, and only the following risk factors are considered as well-established: age, ethnicity , hereditIn 2007, the World Cancer Research Fund (WCRF) and the American Institute for Cancer Research (AICR) published the Second Expert Report for cancer prevention, replacing the First Expert Report conducted in 1997 ,14. ThisSince 2007, several studies have evaluated the association of the recommendations of the Second Expert Report and cancer mortality , as wellA population-based case-control study was developed to evaluate the association between lifestyles and PCa among Spanish men (CAPLIFE study). It was carried out at in two main University Hospitals in Granada (Spain): Virgen de las Nieves and San Cecilio Hospitals and their catchment area. They belong to Metropolitan Granada District, which covers more than 645,000 inhabitants. All those PCa cases that belonged to the aforementioned hospitals were invited to participate by their urologist after being diagnosed of PCa. Study participants were enrolled from May 2017 to December 2019.Ethical Approval for this study was provided by the Ethics Committee of Biomedical Research of Andalusia in March 2017. All men included in the study were fully informed about the study objectives and they signed a written informed consent before their voluntary participation. Confidentiality of data is secured by removing personal identifiers in the dataset.http://cancergranada.org/es/index.cfm).Eligible cases were required to meet the following criteria: (1) new diagnosis of PCa   with hisData were collected between 2017 and 2019 by face-to-face interviews conducted by trained interviewers using a structured computerized epidemiological questionnaire. The same interviewers surveyed cases and controls. Information on the following background variables, for both cases and controls, was obtained: sociodemographic data , lifestyles , and personal/family medical history, including first-degree family history of PCa in father and/or brothers, among other variables. Waist and hip circumference were measured at the interview, while height and weight one year prior to diagnosis were self-reported, and body mass index (BMI) calculated. If there were any missing data, it was recovered by reviewing the clinical record or via telephone contact.The physical activity information was collected using the International Physical Activity Questionnaire (IPAQ), validated for the Spanish population . In addiThe 2018 WCRF/AICR Cancer Prevention Recommendations includes a total of ten recommendations: (1) body fatness, (2) physical activity, (3) consumption of whole grains, vegetables, fruit, and beans, (4) fast food and other processed foods, (5) red and processed meat, (6) sugar-sweetened drinks, and (7) alcohol, (8) dietary supplements, (9) breastfeeding, and (10) cancer survivors’ recommendations. In this study, we omitted the latest three recommendations. The reasons for avoiding the three last recommendations were the following: (i) The use of dietary supplements for cancer prevention and choice to consume nutrients through food alone is largely addressed through the other five dietary recommendations, as AICR refers in the article on the operationalizing of the score ; and  Tercile 1: minimal compliance with the recommendations; (ii) Tercile 2: intermediate compliance and; (iii) Tercile 3: minimum compliance.The Gleason score was collected from the pathological report of each participant. It assigns two grades for each patient: a primary grade is given to describe the cells that make up the largest area of the tumor and a secondary grade is given to describe the degree of differentiation of the cells of the next largest area. Both grades use values from 1 to 5: 1 for a high degree of differentiation and 5 for a minimal degree of differentiation . The aggCharacteristics of the participants were examined using means and standard deviations (SD) for continuous variables and percentages for categorical variables. These characteristics were described for case and control groups as well as 2018 WCRF/AICR terciles for the control group. Chi-squared tests were used to evaluate the level of significance of the differences observed in categorical variables, and Student’s t-tests or one-way ANOVA for continuous variables.p < 0.05. Statistical analyses were performed using statistical program Stata v.15 .Multivariable logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (95% CI) for the association between the 2018 WCRF/AICR recommendations and PCa. The 2018 WCRF/AICR score was analyzed as a continuous variable (one-unit increment) and as a categorical variable through the terciles. The first tercile was used as the reference category (minimum compliance with the cancer prevention recommendations). We used the information from previous studies and directed acyclic graph (DAG) to identify potential confounders; thus, epidemiological and statistical criteria were used to construct the models. An adjusted model was constructed including as covariates: age, educational level, smoking status, primary family history of PCa, and total energy intake. In addition, analyses were also conducted stratifying by aggressiveness: low aggressiveness and high aggressiveness. We estimated the individual association of each component of the 2018 WCRF/AICR score with PCa risk, after adjustment for all other components of the score and the aforementioned potential confounders. For this analysis, participants without information on anthropometric measures, physical activity, or dietary information were excluded. People with implausible energy intake values were also excluded for the analysis. All statistical tests were two-sided and statistical significance was set at Distribution characteristics between cases and controls are shown in p < 0.01).The distribution of controls’ characteristics according to terciles of compliance with the score is provided in p-value = 0.02). Its protective association is maintained when the 2018 WCRF/AICR score is considered as an ordinal variable in terciles, although without reaching statistical significance. The results are similar when we stratify by aggressiveness, aOR = 0.79 (95% CI 0.66–0.95) and aOR = 0.86 (95% CI 0.69–1.06) for each unit of increase in the score for low and high aggressiveness, respectively.When we analyzed the association with recommendation compliance, a slight protective relationship was observed between a higher compliance with the 2018 WCRF/AICR recommendations and PCa when this variable was analyzed as continuous . For eacp-trend < 0.05). Also, not consuming alcohol showed a protective association, aOR = 0.49 (95% CI 0.27–0.91) for men who met with the recommendations (p-trend < 0.05). Results according to aggressiveness are detailed in The mutually aOR for the individual components of the 2018 WCRF/AICR score and PCa are shown in This case-control study explores the association between the 2018 WCRF/AICR Cancer Prevention Recommendations and the risk of PCa, being one of the first studies in exploring this issue. A slight protective association was found between each one-unit increase of the 2018 WCRF/AICR score and the risk of PCa, especially for low aggressiveness PCa. In addition, three components of the 2018 WCRF/AICR score were independently associated with a lower risk of PCa: (i) “Fast foods” and other processed foods high in fat, starches or sugars; (ii) sugar-sweetened drinks; and (iii) alcohol.If we compare the 2018 WCRF/AICR score with the previous recommendations, six of them partially coincide, while the following four recommendations have changed substantially: physical activity, alcohol intake, “fast foods” and other processed foods, and sugar-sweetened drinks . In partTo date the association between compliance with the 2018 WCRF/AICR recommendations and PCa has not been evaluated, but it has already been explored for the 2007 WCRF/AICR recommendations. The studies that use the previous recommendations do not find an association between these recommendations and PCa when the aggressiveness of the tumor is not considered ,23,29,31By stratifying for aggressiveness, a protective association between the cancer prevention recommendations and low aggressiveness PCa was observed; a finding not previously found by other researchers. For cases with a tumor with high aggressiveness, we also observed a protective effect, although the results do not reach the statistical significance. This may be due to the small sample size for high aggressiveness PCa in our study. In fact, other studies find similar results, MCC-Spain , where aRegarding limiting consumption of “fast foods” and other processed foods high in fat, starches or sugars, a recent cohort study, published in 2018, studied the association between “fast foods” and processed food and PCa risk . This stFor sugar-sweetened drinks and PCa, the results are also limited. According to a meta-analysis carried out with four observational studies conducted before 2012, the results are highly heterogeneous and they did not find an association between carbonated beverages and PCa . In our We found that PCa cases without a WCRF/AICR score had a lower educational level , as subjRegarding advantages of our study, we have to point out the use of a previously validated FFQ for the Spanish population, including regional products . In ordeIn conclusion, in this Spanish population-based case-control study, a slight protective association was found between compliance with the 2018 World Cancer Research Fund/American Institute for Cancer Research (WCRC/AICR) cancer prevention recommendations on diet, physical activity, and body fatness and overall PCa, especially in low aggressiveness PCa. In addition, limiting consumption of “fast foods” and other processed foods high in fat, starches or sugars, sugar-sweetened drinks, and alcohol were independently associated with lower risk of PCa. Altogether, it is advisable to comply with healthy lifestyles in the prevention of PCa.


Additionally, motor performance tests and parents’ reports were applied in order to test the reliability and validity of the SFST. Results: The study group had significantly greater thumb-finger test scores—total time in the dominant hand (p = 0.035) and the SD of the touch time in both dominant (p = 0.036) and non-dominant (p = 0.032) hands. Motor performance tests were not correlated with the SFST. Test–retest reliability in 10 healthy children was found for the SD of touch time in the dominant hand . Conclusions: The SFST was successful in assessing the movement pattern variability reported in children with motor difficulties. This exploratory study indicates that the low-cost SFST could be utilized as an objective measure for the assessment of proprioception components, which currently are overlooked by standardized motor performance assessments.Background: Coordination is crucial for motor function, yet objective clinical evaluations are limited. We therefore developed and tested the reliability and validity of a low-cost sensorized evaluation of a rhythmic finger task. Methods: Children with coordination difficulties ( The proprioceptive system provides awareness of the body′s movement and posture through receptors found in the muscles, tendons, and joints . Its funChildren with motor coordination difficulties encounter challenges in participating in various activities at school, home, and in the community ,6, and pA comprehensive evaluation of proprioception is important in order to devise an optimal intervention for children with difficulties in motor coordination . Ayres fThere is, therefore, a paucity of precise and objective tools that evaluate proprioceptive function as part of a standardized clinical assessment ,17. SuchTherefore, in order to accurately assess proprioception, we designed and built an objective low-cost system for quantification of the proprioception components of motor sequencing and timing, the Sensorized Finger Sequencing Test (SFST). We chose to automate the clinical sequential finger tapping test, since this test is widely used as a proprioception marker e.g., ,23). We . We 23])This was a cross-sectional exploratory study. The study was approved by the Institutional Review Board and registered in Clinical Trials.gov . All parents provided a signed informed consent form and all children provided verbal assent for participation in the study.n = 25), and TD children comprised the control group (n = 25). Inclusion criteria were: a final score in the Developmental Coordination Disorder Questionnaire 2007 (DCDQ′07) of ≤46 for the study group and ≥47 for the control group [t-test; Fifty children aged from 5 to 7 years were recruited via a convenience and snowball sampling method, between December 2017 and July 2018. Children who were referred to a child developmental center due to various motor difficulties comprised the study group  : a standThe Beery–Buktenica Developmental Test of Visual-Motor Integration 5th Edition (Beery VMI) : a standThe Sensory Processing Measure (SPM) home form : a standThe Participation in Childhood Occupations Questionnaire (PICO-Q) . This isFor construct validity testing using the known group and discriminant validity procedures, the following four assessments were conducted:The study outline is depicted in For the test–retest, the SFST was administered twice, two weeks apart by 10 control participants, since performance variability characterizes individuals with motor coordination difficulty .The SFST data were analyzed using a LabVIEW code , which was written for this study. Three final scores for each hand were used: (i) the number of in-order touches (until a sequence error emerged); (ii) the total test duration; and (iii) the SD of touch duration for all detected touches of each hand.t-tests for two independent samples or Mann–Whitney tests, according to the data distributions. Categorical variables were summarized according to number and percentage, and groups were compared with Chi-squared tests. Spearman′s or Pearson correlation coefficients were calculated between pairs of continuous variables. All statistical tests were two-sided and tested at a 5% level of significance. Since this was an exploratory study, no alpha correction was applied.The statistical analyses were performed using SPSS version 25. One-way analysis of variance (ANOVA) was used to examine the differences between the three sequences of assessments applied. Shapiro–Wilk′s test was used to examine the distribution nature of all variables. Continuous variables were summarized by means and SDs, or medians and inter-range quartiles (IRQ) and groups were compared with p < 0.001). All of the study group participants scored below the cutoff that indicates DCD or suspected DCD.Since two children refused to wear the SFST, 48 participants (24 in each group) were included in the analyses. The DCDQ′07 final score of the control group was significantly higher than the final score of the study group, with a mean score (SD) of 65.3 (7.2) and 39.5 (4.9), respectively (t(46) = 14.453, p = 0.262); MABC-2-ball skills  = 0.861, p = 0.429); and total time of the SFST in the dominant hand  = 1.054, p = 0.357). This finding confirms no attention or fatigue bias.There were no statistically significant differences between the three sequences applied for the following assessment scores: Beery VMI  = 1.378, Statistically significant group differences were found in all assessments tested, demonstrating a lower performance in the study group .The known group procedure: The control group performed the trial faster with the dominant hand while demonstrating lower SD touch time in both hands, compared to the study group (dy group . In addidy group .Discriminant (or divergent) validity: no statistically significant correlations were found between the SFST and the assessments scores of the Beery VMI, MABC-2, SPM and PICO-Q, demonstrating the discriminant type of construct validity, indicating that the SFST tests a different construct than each of these assessments.p < 0.01; A statistically significant, positive, and strong correlation was found between the two measurements, only for the SD touch time of the dominant hand , which we isolated as a discriminating parameter between children with and without coordination difficulties, was also found to be consistent between test and repeated test for the dominant hand, and thus is a reliable measure of the SFST. However, the total time  was reduced in the second measurement. This may suggest a learning effect that elicits improvement in the performance accuracy through implicit learning  ,31. HoweIn this study, completing the SFST took longer for the children with coordination difficulties compared to the control group. Taking longer to complete a task might be a consequence of their difficulty in correcting errors during motor tasks due to their impairment . InteresThis is an exploratory study, testing a newly developed device to ameliorate the proprioception components  assessment in children. Yet, some limitations should be considered, such as sample size and potential selection bias based on the recruitment method, missing data, and the number of repetitions in the task performance. Further studies should expand this exploration, comparing eyes open versus closed performance, in order to discern whether the between-group differences stem from deficits in proprioception (and then we expect no between-group differences in the performance with open eyes) or from motor control deficit in keeping a rhythm .This study lays the foundation for the clinical use and future development and studies of the low-cost SFST, for the clinical assessment of proprioception in particular, and may contribute to the evaluation of motor coordination difficulties in general. Since we found no correlation with the performance-based assessments that are considered to test proprioception as well, the possible potential of the SFST to ameliorate the current evaluation state of performance by contributing an objective measure of proprioception components should be investigated in future research.


Generalized anxiety disorder (GAD) is a persistent and common mental disorder that entails significant impairments in role functioning and quality of life. Currently available effective interventions include psychological therapies, self-help approaches, and pharmacological treatments, which do not quite meet clinical needs, and the ideal anxiolytic is still being sought. Shu-gan-qing-re (SGQR) formula, a Chinese patent medicine, has been well received by patients with GAD in Chinese clinical practice for years. The present prospective, double-blind, double-dummy, randomized controlled trial is designed to investigate the efficacy and safety of SGQR formula for GAD.A total of 200 eligible participants will be recruited from four hospitals in different parts of China. They will be randomly assigned to either the study group or the control group in a ratio of 1:1. Participants allocated to the study group will receive SGQR formula and buspirone placebo, while buspirone and SGQR placebo will be applied in the control group. Six scheduled visits will be conducted over the course of 8 weeks. Outcome measurements include Hamilton Anxiety Rating Scale (HAM-A), Hamilton Depression Rating Scale-17 (HAMD-17), Clinical Global Impression-Improvement Scale (CGI-I), Traditional Chinese Medicine Syndrome Scale for GAD, and pro-inflammatory cytokine tests: interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha. Adverse reactions will be evaluated by using the Treatment Emergent Symptom Scale (TESS). Safety outcomes and adverse events will also be recorded.The study will provide scientific and objective assessments for the efficacy and safety of SGQR formula for patients with GAD, hopefully offering clinicians an alternative approach to GAD.ChiCTR-IPR-17013058. Registered on October 20, 2017.Chinese Clinical Trial Registry, ID:  Generalized anxiety disorder (GAD) is a persistent and common disorder characterized by feelings of threat, restlessness, irritability, sleep disturbance, tension, and symptoms such as palpitations, dry mouth, and sweating . An epidGAD has a relapsing course, and treatments rarely result in complete resolution of symptoms. In the short to medium term, effective intervention includes psychological therapies, such as cognitive behavioral therapy; self-help approaches based on cognitive behavioral therapy principles; and pharmacological treatments . Until tTraditional Chinese medicine (TCM) is the most popular alternative and complementary medicine in China. It has a long history in the treatment of mental disorders and has favorable clinical effectiveness. According to the theory of TCM, anxiety results mainly from the stagnation of the liver qi, and inner heat emerges as a result. Shu-gan-qing-re (SGQR) formula is tailored from famous TCM prescriptions: Xiao-yao powder and Gan-mai-da-zao decoction. It is composed of Radix Paeoniae Alba, Radix Bupleuri, Angelica Sinensis, and seven other herbals and functions by smoothing the liver qi. SGQR formula can help to relieve anxiety and has been used in Chinese clinical practice for years.The present multi-center, prospective, double-blind, double-dummy, randomized controlled trial is designed to investigate the efficacy and safety of SGQR formula, setting buspirone as the control treatment. We hope that, through this rigorously designed study, we can provide scientific and objective assessments for the efficacy and safety of SGQR formula for patients with GAD and possibly offer the world an alternative anxiolytic.This is a multi-center, prospective, double-blind, double-dummy, randomized controlled trial. The flowchart of the study is presented in Fig. The clinical trial has been registered on the Chinese Clinical Trial Registry (ChiCTR-IPR-17013058). The study protocol (version 2.0 in August 2017) has been approved by the research ethics committee of Beijing Anding Hospital, Capital Medical University ((2017)63-201772FS-2). The research ethics committee will also be responsible for supervising all procedures of the study, including participant recruitment, randomization, conduction, and data storage. In case of any changes to the study protocol, we will submit a written application to the research ethics committee. They will decide whether it is acceptable to make the change.A total of 200 eligible participants will be recruited from four hospitals in different parts of China: Beijing Anding Hospital, Xiamen Xianyue Hospital, Tangshan 5th Hospital, and Zhumadian 2nd People’s Hospital. Both inpatients and outpatients will be screened for participation from November 2017.Potentially eligible patients will be invited to take part in our study. Prior to enrollment, researchers will fully introduce the detailed procedures of the study to the patients. All questions and concerns raised about the study will be addressed at length. Patients will also be informed of the probable benefits and potential risks and assured that participation is entirely voluntary. Patients who meet all of the inclusion criteria and none of the exclusion criteria will be enrolled after providing written informed consent. The personal information of all the participants will always be kept confidential.Participants meeting all of the following criteria will be enrolled: (1) inpatients or outpatients with GAD diagnosed in accordance with the International Classification of Disease-10; (2) those with a diagnosed TCM pattern of liver-qi stagnation transforming into fire, which usually manifests as irritability, constriction in the chest, hypochondriac distention, gastric discomfort with acid regurgitation, dry mouth or bitter taste in the mouth (or both), constipation, headache, conjunctival congestion, tinnitus, reddened tongue with yellow fur, and rapid pulse; (3) ages between 18 and 65 (no limitation on gender); (4) Hamilton Anxiety Rating Scale (HAM-A) scores between 14 and 39 (inclusive) at screening and baseline; and (5) written informed consent obtained either personally or by proxy.Participants meeting any one of the exclusive criteria will not be enrolled: (1) patients with serious suicidal tendencies; (2) patients with glaucoma; with severe or unstable internal medical conditions involving mainly the heart, liver, kidney, endocrine system, or the blood; or with diseases that may induce acute or chronic abnormal levels of pro-inflammatory cytokines ; (3) history of epilepsy (hyperpyretic convulsion in the childhood excepted); (4) alcohol or drug dependence in the previous year; (5) anxiety resulting from other mental or physical disorders; (6) pregnant or breastfeeding women or women who plan to conceive during the study period; (7) history of severe drug allergy or allergy to buspirone; (8) participation in other clinical drug trials in the previous 30 days; (9) intake of monoamine oxidase inhibitor or buspirone in the previous 4 weeks; (10) regular medicine intake cannot be guaranteed because of being uncared or other reasons; (11) a reduction of the HAM-A score of at least 25% at baseline compared with the screening time; and (12) a Hamilton Depression Rating Scale-17 (HAMD-17) score of more than 7 at screening.Eligible participants will be randomly assigned to either the study group or the control group in a ratio of 1:1. A computer-generated random allocation sequence will be produced by an independent statistician using SAS software . Based on the allocation sequence, drug packages will be labeled serially. Participants will receive the corresponding drug package in the order of recruitment. The complete random sequence will be kept by the primary researcher in Beijing Anding Hospital. Emergency envelopes containing the random allocation individually will be prepared for each center, which can be unsealed only in the case of serious adverse events.Pharmacists in charge of labeling the drug packages do not take part in the conduction of the trial. Participants and researchers responsible for screening, recruitment, dispensing medicine, and outcome assessment will all be blind to the group allocation. Statistical analysis will be conducted by independent statisticians who will know only the group codes but not the corresponding interventions.Participants allocated to the study group will receive SGQR formula and buspirone placebo, while buspirone and SGQR placebo will be applied in the control group. The drug and its matching placebo will have a similar appearance, weight, and taste. SGQR formula and placebos will be provided by Tiansheng Taifeng Pharm Co. Ltd. , and buspirone will be manufactured by Enhua Pharm Co. Ltd. .Triticum aestivum L 0.86 g, and Fructus Jujubae 0.57 g, 10 herbals in total.SGQR formula is prepared in the form of condensed pills. The pills will be packed at a unit of 4 grams, whose ingredient is equivalent to Radix Paeoniae Alba 1.14 g, Radix Bupleuri 0.86 g, Angelica Sinensis 0.57 g, Radix Curcumae 0.57 g, Poria Cocos 0.69 g, Bulbus Lilii 0.69 g, Cortex Albiziae 0.69 g, Radix Glycyrrhizae 0.34 g, There will be three dosages of drugs: the starting dosage , the low dosage , and the high dosage .In the first week of intervention, participants will be prescribed the starting dosage for the first three days and the low dosage for the remaining four days. From the second week on, the dosage will be adjusted by the researchers. The recommended indicator for doubling to high dosage is a reduction of not more than 25% of the HAM-A score compared with the baseline after the first week’s intervention, keeping low dosage otherwise. Moreover, high dosage and low dosage can be alternated anytime during the intervention period in accordance with patient’s reaction to the previous treatment.Drugs and placebos covering the whole course of treatment for each participant will be packed uniformly and individually. In each center, there will be a pharmacist responsible for dispensing the prescription. At every visit, participants will receive the drugs scheduled for the next intervention period, and the drugs left from the previous period will be returned. The dosage will be explained to the participants orally and in writing and also recorded in a medical booklet. Medication compliance will be calculated accordingly. The scheduled duration of treatment is 8 weeks unless a serious adverse event happens or the participant quits. Non-pharmacologic care is permitted but other psychiatric medication is prohibited. Administration and dosage of concomitant medication for physical conditions will be permitted and recorded in detail.Any question raised by the participants will be answered to promote completion. The detailed study schedule is listed in Fig. Outcome measurements are scheduled at screening, baseline, and the end of weeks 1, 2, 4, and 8.Basic data of all the participants, including name, gender, date of birth, marital status, educational level, date of diagnosis, and medical history, will be collected. General physical examination results, including vital signs , will also be recorded. Concomitant medication will be inquired about and documented at every visit.HAM-A, which is one of the first and most widely used rating scales to measure the severity of anxiety symptoms, will be set as the primary outcome . There aHAMD-17 is set as one of the secondary outcomes because comorbidity of depression and anxiety is rather common in health-care settings . HAMD-17Another secondary outcome focuses on the improvement in overall symptom severity and functioning, measured by using the Clinical Global Impression-Improvement Scale (CGI-I), which consists of a 7-point global rating  . This scThe TCM Syndrome Scale for GAD is a tailored scale for GAD and is based on the theory of TCM. It concentrates more on subjective discomfort in combination with objective clinical signs. Subjective discomfort includes worry without reason, hesitation, being easily frightened, and a disinclination to talk because of a deficiency of qi. These symptoms may not be fatal but always trouble the patient seriously. Objective clinical signs focus on the complexion, color of the lips and tongue, tongue coating, and the pulse. When the patient is evaluated with this scale, the differentiation and severity of the TCM syndrome can be concluded.Pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha, will be tested at baseline and the last visit. An altered cytokine balance  was found in GAD . PreviouSafety outcomes involve complete blood count, electrolyte panel, liver and kidney function tests, urinalysis, urine pregnancy test, and electrocardiograph. Safety outcomes will be assessed at screening time and the last visit.Adverse reactions will be checked by using the Treatment Emergent Symptom Scale (TESS) at every visit . If any Before the conduction of the protocol, a range of training will be provided to all the researchers, ensuring a full understanding and mastery of the standard operating procedures. Any change to the protocol should be reported in writing to the primary researcher in Anding Hospital and can be applied only after permission. The quality of CRFs will be regularly supervised by independent investigators in Anding Hospital. Researchers will be notified in a timely fashion if any error exists.As there is no large randomized controlled trial of SGQR formula for GAD, we referred to the experience of experts in this field. A response rate of 91% for SGQR was assumed. As to buspirone, the response rate was about 75%, according to the literature . In the t test  or non-parametric test  will be used for continuous variables when conducting baseline comparisons. The repeated measures ANOVA  will be used when analyzing the primary and secondary outcomes. Chi-squared or non-parametric test will be applied for categorical variables. The analysis of efficacy will be performed in accordance with the intention-to-treat principle. Missing values will be imputed by the last observation carried forward method. Subgroup analysis will be conducted if necessary. A P value of less than 0.05 will be considered statistically significant. A mixed-effects model for repeated measures will be performed as a sensitivity analysis of the primary efficacy outcome.CRFs will be entered by two independent researchers and supervised by a third one to ensure accuracy. Data analysis will be performed by statisticians who are blind to the randomization by using SPSS  Statistics for Windows, version 20.0 . Continuous variables will be presented as mean and standard deviation, and categorical variables will be presented as frequency or percentage. Demographic data and outcome measurements at baseline will be listed to ensure relatively even baseline levels. A Mid-term date analysis will be conducted after half of the recruitment is finished, and the principal investigator will decide whether any measure should be taken in light of the results. All of the data will be deposited in the platform developed by our team. Anding Hospital will be responsible for the data, and other researchers can have access to the data only after application and permission.Just like many other Chinese patent medicines, SGQR formula has been widely used in China for years, but strong clinical evidence supporting its use is still lacking, limiting its global popularization. Buspirone, a 5-hydroxytryptamine 1A receptor agonist belonging to azapirones, has been approved for the treatment of GAD in adults and has been shown to be effective , 18. A CIn this study, we chose the commonly used buspirone as the control and set a number of scientific and objective assessments. We hope that the results of this clinical trial, if successfully conducted, will provide a promising approach to treating GAD.The protocol version was 2.0 (August 2017). Participant recruitment began in January 2018, is ongoing at the time of this writing, and will be finished by about June 2020.