Because many elements of a woman’s life, such as her upbringing or career, occur at different points in her life, some may be risk factors for developing problem drinking only at certain periods during the life cycle. Few long-term studies have focused on risk factors throughout the life span, leaving scientists to glean what factors they can from studies of specific age groups of women. Clearly, not all women in a given population are equally vulnerable to becoming problem drinkers. Certain behaviors or elements of a woman’s history and genetic makeup may increase her risk of developing a harmful drinking pattern. These elements are called risk factors for problem drinking.What is meant by risk factors? Ideally, with longitudinal research, which follows a group of subjects over a period of time e.g., , it woulSome risk factors apparently are present throughout the female life span. Others are specific to certain periods of life, including the adolescent, young adult, middle- age, and older stages. Because longitudinal followup studies of women are scarce, researchers must cull the published literature for risk factors cited at each stage of the life span. Such factors are “markers” of increased vulnerability; that is, they often clearly are antecedent but sometimes only are correlated with the development of problem drinking and cannot definitively be called risk factors. A woman’s role in society may be a risk factor, and role is defined for both men and women primarily by age, gender, socioeconomic class, and ethnicity.This article describes those risk factors that appear to exist for various age groups among women to increase awareness among people involved in the alcohol field about the critical role of stages in the life span. Efforts to prevent problem drinking should target women in each age group (described below) who manifest risk factors.Why examine life stages? Issues that may be relevant to prevention and treatment of adolescents and young women may be irrelevant to prevention and treatment directed toward older women. Alcohol use and the consequences of use also appear to shift among age groups. For example, when alcoholism treatment began a half century ago, typical patients seen in treatment facilities were in their forties. For some decades now, typical patients have been younger. Of those in treatment in the United States, 35 percent are between the ages of 25 and 34, and 25 percent are between the ages of 35 and 44 . These fOne obvious risk factor that is important in predicting a greater likelihood of problem drinking among both women and men is a family history of problem drinking . StudiesIn all stages throughout the life span, women consistently report the significance of the social context in which they drink. Among adolescents, data suggest that girls are more strongly influenced by peer drinking than are boys  and thatTransmission of these drinking patterns from significant others to the women must be inferred; it is reported frequently that more females than males with alcohol abuse or dependence have spouses or significant others who are problem drinkers . The impAt all stages over the life span, female problem drinking is linked to depression. More than 20 years ago, Support for a linkage between female problem drinking and depression comes from both epidemiological and clinical sources. Over the years, the link between female problem drinking and depression has been reported persistently in the literature . AntecedFemale problem drinkers who report preexisting depression differ from primary problem drinkers in several ways. They less often have a positive family history of problem drinking, have a shorter history of problem drinking, and have a more favorable prognosis .Another risk factor encountered throughout a woman’s life is her reactions to stressful events and her ability to cope with these circumstances. Stress, distress, and coping are pivotal in women’s development of problem drinking. Stress involves life events that impact a person negatively; distress is a negative response, usually in response to stress; and coping styles are used to reduce stress.Some researchers posit that stressful events, such as trauma, are antecedent to women’s problem drinking, and some psychological theorists hypothesize that coping strategies used to modify the effects of stress may include drinking as well as seeking help and blaming others . CertainThe distinction between the effects of stress, which is a life event, and distress, which is an affective response, appears in a study comparing female problem drinkers in treatment and their age peers without drinking problems a. The twEarly reports in the literature emphasized specific traumatic events as precipitating abusive drinking. In clinical treatment settings, alcoholic women were more likely to cite the influence of traumatic events  than were alcoholic men. However, the interpretation of such reports, which label the traumatic event as the precipitant of drinking, has been criticized on two grounds: the terms are vague, and the reported link between unhappy life events and heavy drinking might have come from studies of a subgroup of sociopathic women for whom sociopathy produces both heavy drinking and negative events .Another stressful life event that has been connected with women’s problem drinking is childhood sexual or physical abuse. Although presenting a history of abuse as the sole or primary etiology of female problem drinking is difficult, evidence is accumulating that indicates its significance . ChildhoOne line of inquiry for researchers in this field that may further define the relationship between early life experience and problem drinking could be directed toward parental neglect and inadequate supervision in alcoholic families . A revieAlthough there is a vast amount of literature on adolescents’ use and abuse of alcohol, considerably less is known about adolescent girls than about adolescent boys. However, studies comparing the two groups have been increasing e.g., , as haveThe most recent results available from surveys of 8th, 10th, and 12th graders ; table 1Early adolescence differs psychologically and socially from late adolescence. This is important to consider when reviewing the environmental forces that influence adolescent drinking. For example, in early adolescence, parental influence and parents as role models may be more important than they are in late adolescence. As a person moves through adolescence, the role and influence of peers become more significant ; table 2strong evidence for the primacy of susceptibility over exposure  in causing alcohol misuse among young adolescents. This appears to be more true for boys than for girls. . .young adolescent girls misuse alcohol because of both individual susceptibility and group exposure (p. 8).Many risk factors emerge from the literature for adolescent problem drinking, but no particular one appears to be typical of this problem. Instead, it appears that the more risk factors manifested by the adolescent, the more likely he or she is to engage in problem drinking .One risk factor that appears most relevant for adolescent girls is behavior problems, variously described as antisocial or aggressive behavior  or as deFrequent school absences and low educational aspirations have been cited as possible clues to problem drinking among adolescent girls . Poor scA family history positive for alcohol abuse and dependence and a dysfunctional early family environment surface as factors involved specifically in the development of problem drinking among adolescent girls. A dysfunctional family environment is characterized by “heightened marital conflict” between parents and “inadequate parenting” . ConsistDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition  not been identified as alcoholics prior to the loss of their mates. As a result their rapid decline [and the start of their heavy drinking] has often been attributed to the grief and despair of widowhood , p. 168.Even without a history of drinking with a husband, widowhood may produce among some women an increase in alcohol intake for at least the first year after the loss. When those in the study of older alcohol abusers  who wereThis possible risk factor rarely has been studied, but some evidence suggests that retirement has a more negative impact on retired women than on retired men. Women build social networks within their places of employment, and retirement may be a stressor of significant proportions, particularly if they have limited family networks and other interests . AnotherAlthough older men and women report receiving prescriptions for psychoactive drugs to the same extent (70 percent) for both genders, women are heavier users of these drugs—as they are in all age groups . A recenMany residential centers for older individuals do not facilitate drinking. However, some retirement communities, complete with golf courses and other amenities, seem to stimulate drinking and, occasionally, heavy drinking. Although this phenomenon has been described by adolescents at risk for problem drinking, and patterns of distress are more closely tied to female than to male drinking in this age group. Middle age historically has been associated for women experiencing loss, depression, the “empty nest,” and menopause. Although most women in their forties and fifties handle these challenges well, for some, the events of middle age are overwhelming and distressful and may lead to problem drinking. Second, in light of the presence of more than one risk factor for each life stage, it is important to remember that the more risk factors that are present, the greater the likelihood that problem drinking will develop.Two dicta emerge from a review of risk factors over the life span of women. First, although most risk factors characterize all age groups, some are unique to each stage in the life cycle. For example, inadequate control over impulses characterizes Certain questions remain to be answered about alcohol abuse and dependence among women in these age groups. For example, among adolescent girls, are those who exhibit antisocial personality behaviors and depression more likely to be abusing alcohol? Among young adult women, the question of why the gender ratio for risk of alcohol problems converges in the thirties needs exploration. Why is the thirties a decade of such great vulnerability to problem drinking for women? How do aging, depression, and alcohol intake interact among middle-age women? The question of what role distress patterns or feelings play in the etiology of problem drinking in this age group needs further study. Finally, among older women, areas that need investigation include the use of prescribed psychoactive drugs, marital patterns that lead to husbands and wives drinking together, and adaptation to widowhood.


Inductive reasoning can be performed in different contexts, but it is unclear whether the neural mechanism of reasoning performed in a thematic context  is the same as that performed in a taxonomic context . In the present study, participants were required to judge whether a conclusion was acceptable or not based on its premise, for which the taxonomic or thematic distances between premise and conclusion objects were either far or near. The Event related potential (ERP) results indicated that the effect of context (taxonomic vs. thematic) was initially observed in the P2 component; while the distance effect (far vs. near) was observed in N400 and late components. Moreover, the distance effect on thematic-based inductive reasoning was found in the anterior regions, while the distance effect on taxonomic-based inductive reasoning conditions was found in the posterior regions. These results support the view that inductive reasoning is performed differently under different semantic contexts. Inductive reasoning is a complex, high-level cognitive process that is pervasive in human life. It can generate new knowledge based on limited information and extend to new situations. This ability can effectively improve human learning  to context also help us to investigate whether different types of inductive reasoning require different amounts of cognitive resources at different time windows. Previous studies have found that the cognitive demand during taxonomic and thematic judgments may differ depending on whether the object is an artifact such as a hammer or a natural object such as a cherry  participated in the formal experiment, with five excluded due to excessive eye movements. Thus, 24 participants remained in the ERPs. All participants were right-handed with normal or corrected-to-normal vision. All participants provided written informed consent and were monetarily compensated. The experiment was approved by the ethics review board of Jiangxi Normal University.Sixty-four healthy undergraduate students (aged 18–25 years) rated the experimental materials in a pilot test. Another 29 healthy undergraduate students . In the present study, each condition had 128 trials that appeared with same frequency. Four types of conditions appeared randomly to avoid the expectation of subjects. According to previous research, we translated the word pairs into Chinese and selected the artificial words ; 64 pairs had a thematic-near relation, whereby the two words were thematically related with a near distance ; 64 pairs had a taxonomic-far relation, whereby the two words were taxonomically related with a far distance ; and 64 pairs had taxonomic-near relation, whereby the two words were taxonomically related with a near distance .t test indicated that the thematic-near word pairs  were significantly different from the thematic-far word pairs  in terms of thematic relation, t = 12.48, p < 0.01. The taxonomic-near word pairs  differed significantly from the taxonomic-far word pairs  in terms of taxonomic relation, t = 16.78, p < 0.01.Finally, the 64 pairs of words in the four conditions were retained and used in the formal experiment. A paired-The formal experiment was a category-based induction task  were presented sequentially on a computer screen and each argument was repeated twice. The entire formal experiment comprised 512 trials (128 in each condition). As shown in Electrophysiological activity was recorded using a 64-channel EEG system , with the reference electrodes on the left and right mastoids. An electrode placed under the right eye  allowed the monitoring of blinks and vertical eye movements. The impedance of all electrodes was maintained below 5 kΩ. Raw data were band-pass filtered between 0.01 and 100 Hz and digitized at a sampling rate of 500 Hz. Trials with EOG artifacts (a mean EOG voltage exceeding ±80 μV), and those contaminated with artifacts due to amplifier clipping, bursts of electromyographic activity, or peak-to-peak deflections exceeding ±80 μV were excluded from averaging.p-values in the ANOVA were corrected with the Greenhouse-Geisser correction when necessary and multiple comparison was corrected with Bonferroni criterion . Frequencies lower than 0.01 Hz or higher than 30 Hz were digitally filtered (the filter slope 24 dB per octave). The analysis epoch was 1,000 ms with respect to the averaged voltage over the 200-ms epoch before the onset of the conclusion stimulus. The ERP waveforms were time-locked to the onset of the conclusion stimuli. The averaged epoch for the ERPs to the conclusion stimuli, including a 200-ms pre-stimulus baseline, was 1,200 ms. According to visual inspection of the grand average waveforms and previous studies  = 6.92, p < 0.05, η2 = 0.59. There was no main effect of context, F = 1.02, p = 0.321, η2 = 0.038. There was a marginally significant interaction between context and distance, F = 3.16, p = 0.087, η2 = 0.108. The simple effect analysis indicated that frequency of ‘yes’ responses for thematic-far was higher than for taxonomic-far at a marginal significance level (p = 0.065).F = 16.54, p < 0.001, η2 = 0.37], with faster responses in the near distance than in the far distance. Whereas, the main effect of context was not significant . There was no interaction between the context and distance .For the analysis of RT, any outlier (beyond two standards of mean RT) were excluded for each subject. The percentage of trials with outlier RTs was approximately 1.62, 1.27, 1.24, and 1.37% for thematic-near, thematic-far, taxonomic-near, and taxonomic-far, respectively. The repeated measures ANOVA showed a main effect of distance . Simple-effect tests showed that taxonomic relations elicited a larger P2 than the thematic relations in the left (p = 0.002), middle (p < 0.001), and right sites (p < 0.001), implying that the effect of context on P2 amplitude was greater in middle and right sites than left sites.The ERPs evoked by the conclusion in the different conditions are shown in F = 5.02, p = 0.035, η2 = 0.18, and effect of distance, F = 12.57, p = 0.002, η2 = 0.35. Thematic relations elicited a significantly larger N400 amplitude than taxonomic relations, and the far distance generally elicited a larger N400 amplitude than near distance.Difference waves and topographical maps of the distance effect for thematic relation (left) and taxonomic relation (right) are shown in F = 4.27, p = 0.003, η2 = 0.16]. A simple-effect analysis showed that thematic relations elicited a larger N400 amplitude than taxonomic relations in central (p = 0.014) and central-parietal (p = 0.015) areas, while the effect of context was non-significant in other sites . In the same way, a three-way interaction of context, frontality, and laterality was observed . A simple-effect analysis revealed an effect of context at the following sites: FC3, C3, Cz, CPz, Pz, C4, and CP4 , with a larger N400 amplitude for thematic relations than for taxonomic relations.An interaction between context and frontality was also observed . A simple-effect analysis showed that far distance elicited a larger N400 amplitude than near distance conditions at the left (p = 0.005), middle (p = 0.001), and right sites (p = 0.002), implying that the effect of distance on N400 amplitude was greater in the middle and right sites than left sites. At the same time, there was a three-way interaction of context, distance, and frontality . A simple-effect analysis showed a distance effect on thematic relation in the frontal (p = 0.013) and frontal-central (p = 0.011) regions, while the distance effect on taxonomic relations was found in the central-parietal (p = 0.001) and parietal (p = 0.001) regions.There was a two-way interaction of distance and laterality . Simple-effects tests showed that far distance evoked more negative waves than near distance conditions in all regions within each time window , while the effect of distance on the LNC amplitudes was greater in posterior sites than in the anterior sites.To investigate the time course of the LNC more precisely, we analyzed three successive intervals, 500–700 ms window, an interaction of distance and laterality was found . A simple-effect analysis showed that far distance evoked greater negative waves than near distance conditions in the left (p = 0.018), middle (p = 0.002), and right sites (p = 0.003), implying that the effect of distance on LNC amplitude was greater in middle and right sites than left sites.Within the 600The main purpose of this study was to differentiate the electrophysiological response to inductive reasoning under thematic and taxonomic contexts. Behavioral results showed that there was a marginally significant difference of “yes” response between the two contexts when the distance is far; whereas the likelihood of a “yes” response was higher and the RT was shorter for the near distance compared to the far distance, which indicates that the reasoning process between the premise and conclusion required more effort in far distance than near distance conditions , but no effect of distance (near vs. far). During the N400 time window, both the context effect and distance effect were observed. In the LNC time window, only a distance effect was observed.The difference between the two contexts of inductive reasoning were initially observed in the P2 time window. Taxonomic-based inductive reasoning elicited a larger P2 amplitude than thematic-based inductive reasoning trials. The P2 is generally associated with perceptual decoding  and distance effect (near vs. far) were observed. This indicates that, after decoding the related feature of two categories (premise and conclusion) within the P2 time window, participants made an elaborative semantic integration of the relation between premise object and conclusion object, and determined whether the conclusion object had the same property as the premise object. Interestingly, the P2 amplitude is larger for taxonomic than thematic relations while the opposite is true for the N400 amplitude. This implies that the thematic relation is not intensively processed in the earlier time window  but is processed in the later time window, in which the knowledge-based relation is retrieved and integrated.The N400 component is typically related to semantic integration  regions, while distance effect on taxonomic-based reasoning was observed in the posterior  region. Previous studies have shown that inductive reasoning mainly involves the left medial frontal or the left frontal gyrus (Goel and Dolan, In conclusion, a modified inductive reasoning task was used to investigate the electrophysiological difference between thematic and taxonomic-based inductive reasoning. ERP results revealed a significant effect of distance on both types of reasoning during the N400 time window, but the scalp distribution of this distance effect was different between these two types of semantic processing, with an anterior distribution for thematic-based reasoning and a posterior distribution for taxonomic-based reasoning. This supports the view that these two types of semantic processing may recruit different neural networks.One possible confound for our results is that different sentences were used in the different conditions. People may be able to perceive the sentences in one of the conditions more easily and less easily in other condition. Though a large number of items were used in each condition, reducing the cause for concern. In future studies, the same sentence should be used as the “second sentence” across conditions to completely exclude this confound.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The experiment was approved by the ethics review board of Jiangxi Normal University.FLi and LZ contributed to the conception and design of the study. FLiu organized the database and performed the statistical analysis and wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


More than 50 percent of women in the United States drink alcohol. In general, they abstain more and drink less frequently than men. Women’s drinking behavior is determined by demographic characteristics, such as age, ethnicity, employment, and marital status, as well as by individual and environmental risk factors. Gender is a strong predictor of drinking behavior. According to a 1990 national survey of drinking by American adults , men areData from 39 longitudinal surveysDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition [American Psychiatric Association so variable across studies remains to be determined.The gender differences in drinking and heavy drinking rates are paralleled by gender differences in the prevalence of the clinical diagnoses of alcohol abuse and alcohol dependence.  affect the drinking behavior of both women and men.In this article, we review general trends in women’s alcohol consumption over the last 20 years and present data from a 10-year longitudinal study conducted between 1981 and 1991. We describe the influences of some demographic variables on women’s drinking and identify personal and social risk factors for problem drinking.Not only have drinking patterns among women varied across different periods in history, but society’s concern about women’s drinking has varied even more . During To test the notion that women’s drinking levels were converging on those of men over the last two decades, In an analysis of a 1983 and a 1988 national survey, Because Hilton’s and Williams and DeBakey’s studies used somewhat different criteria to assess drinking and cannot be compared directly, we reanalyzed the 13 surveys used in their studies, two 1990 surveys, and our own 1991 national survey. The data  suggest In addition to looking at time trends of drinking behavior in the general female population, we analyzed changes in drinking patterns and drinking-related problems of individual women over a period of 10 years . For thiA comparison of results from the 1981 and 1986 surveys indicates that individual drinking behavior fluctuates significantly over time . This caThe greatest changes in drinking behavior were found among women in the youngest age group (21 to 34 years). A comparison of data from the 1981 and 1991 surveys showed modest declines in most measures of drinking behavior. , can explain individual drinking patterns.Like gender, age is a strong predictor of drinking behavior. Numerous surveys have shown that younger women have higher rates of heavy drinking and alcohol-related problems than older women see  and 2. FThese studies also suggest that younger and older women have different styles of drinking. Younger women tend to engage in heavy episodic drinking that may lead to severe adverse behavioral or social consequences. These can include alcohol-impaired driving and alcohol-related car crashes, both of which have shown dramatic recent increases among younger women .Drinking in older women, however, seems to be characterized by frequent light or moderate drinking. In the 1984 survey, for example, daily drinking was at least as common among women drinkers over 60 as among drinkers in other age groups . A 1989 Various surveys have shown associations between a woman’s marital status and her drinking behavior e.g., . These sAlthough in most surveys heavy drinking and drinking-related problems are more prevalent among women who are divorced or separated than among married women , two stuA few longitudinal studies have tried to determine whether women’s drinking is a cause, consequence, or accompaniment of divorce and separation. Depending on the individual situation, a woman’s drinking probably can be any one of these: an analysis of divorce rates and alcohol consumption in the United States from 1933 to 1984 concluded that divorce rates influence alcohol consumption but not the reverse . SimilarData from our 1981/1986 longitudinal study indicate that the relationship between divorce and drinking depends on a woman’s history of problem drinking . For womFor women who are problem drinkers, however, drinking is a risk factor for subsequent divorce or separation. Women reporting heavy drinking or problem drinking in 1981 were more likely to be divorced or separated in 1986 than were nonproblem drinkers. Somewhat surprisingly, partnership dissolution led to a reduction in problem drinking for these women, perhaps by removing the woman from a distressing relationship in which partnership problems and excessive drinking were mutually reinforcing .Another marital status that is strongly associated with women’s drinking and drinking-related problems is cohabitation. In our 1981 national survey, cohabiting women reported the highest percentage of drinking (100 percent), heavy drinking (18 percent), intoxication, problem consequences, and alcohol dependence symptoms . A similDuring the 1970’s, employment outside the home was considered to affect women’s mental health and drinking behavior adversely, especially when combined with marital or family roles . FindingRecent survey data are consistent with a new hypothesis that multiple roles, such as family, marriage, and paid employment, can be beneficial for women, reducing their risks of both mental health and drinking problems . PotentiWhether employment has beneficial or adverse effects on a woman’s drinking behavior depends in part on the kind of job she has. In several recent studies, women working in nontraditional, male-dominated occupations reported increased drinking and/ or adverse drinking consequences. For instance, The influence of gender composition of coworkers on drinking behavior seems to be specific for women. Men’s drinking behavior does not seem to be affected by this factor .Several interpretations of the relationship between employment that is nontraditional to women and increased drinking by women have been suggested. These include peer influences , more drinking opportunities in nontraditional employment settings, stress related to a minority status in male-dominated occupations, or the use of drinking as a symbolic expression of power and gender equality .not to have. Data from our 1981/1986 survey show that an unwanted status  can increase the risk of problem drinking in women. An unwanted status is associated with increased alcohol consumption and more adverse drinking consequences. This is especially true for drinking women who had access to alcohol in their home  reported that they felt less sexually inhibited after drinking . These eSome surveys see  have sugAnalysis of women’s drinking over the past 20 years has suggested a moderate increase in alcohol consumption during the 1970’s, followed by a slight decline in the 1980’s . DrinkinThe decline in women’s drinking noted so far seems to be somewhat more gradual or less consistent than the concurrent decline in alcohol sales . FurtherSimilar attention must be paid to the changes in drinking patterns among minority women. So far, the trend toward reduced alcohol consumption seems to be clearest for white women and less pronounced among African-Americans and Hispanics . Future The overall decline in women’s drinking should not lead to alcohol research shifting away from women’s issues for at least two reasons. First, there are some indications that drinking problems and alcohol disorders may still be increasing in some age groups. Future research must determine whether the higher rates of alcohol problems among younger women found in some studies e.g.,  are a reSecond, if generally decreasing alcohol consumption leads to a shift in drinking norms and reduced tolerance toward people with alcohol-related problems , it coulDrinking behavior in women clearly is related to demographic characteristics, such as age, marital status, and employment status. Therefore, future prevention and treatment research should focus on specific subgroups as well as the female population in general. Recent research already has identified and addressed some subgroup-specific characteristics of drinking behavior. More detailed studies are needed to answer questions such as the following and to translate the findings into new prevention strategies:How can heavy episodic drinking and alcohol-impaired driving be reduced in young women?How do divorce and separation affect women’s drinking behavior? When do they have beneficial effects and when do they have adverse effects?How can the apparent adverse effects of cohabitation on drinking behavior be prevented or addressed?When are multiple social roles harmful or protective for women’s drinking behavior?What effect does the lack of social roles have on drinking? How can this lack be counteracted?The answers to these questions can lead to prevention and treatment approaches that target specifically the shared or distinct characteristics of both women’s and men’s drinking behavior and thus improve the effectiveness of intervention for both genders.


The ileal pouch anal anastomosis (IPAA) was introduced in 1978 by Parks and Nicholls. The procedure was designed for patients with ulcerative colitis, for whom a total proctocolectomy was indicated, as an alternative to a permanent ileostomy [Short-term morbidity after IPAA ranges from 28 to 58% and failure is due to technical or inflammatory complications. Remzi et al. have published the largest series on redo pouch surgery [Redo bowel surgery is not easy .This is particularly evident in the pelvis. Pouch excision carries a high rate of short- and long-term complications, 57% and 37%, respectively [The literature on minimally invasive approaches for redo IPAA is very limited. In 2016, Chadi et al. submitted a presentation to the annual Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) meeting with a case series of seven patients undergoing laparoscopic revision of their pouch with two patients having laparoscopic pouch excision.The novel technique of transanal total mesorectal excision (taTME) was originally described by Sylla et al. . This teWe present a case of an adult female with a long history of UC, refractory to medical therapy who underwent a laparoscopic total proctocolectomy and J-pouch reconstruction at our center. Unfortunately after the initial discharge from hospital, the patient required multiple admissions for pelvic sepsis. Ultimately, she was re-referred internally for a second opinion after the clear evidence of J-pouch failure secondary to an anastomotic leak. During the last admission, it was decided to bring her to the operating room, semi-electively for a TAMIS J-pouch excision. Prior to the procedure she was optimized medically.®. The final step was to re-model the diverting loop ileostomy (see video link).The operation was started transanally, using the TAMIS platform. The anastomosis was carefully dismantled, followed by cephalad dissection of the pouch. Specific attention to plane identification was observed. After most of the dissection was completed from the bottom up, a second team joined for laparoscopic assistance, sharp dissection was conducted and eventually the rendezvous occurred. The pouch was completely excised from the pelvis. A Pfannenstiel incision was created and the pouch was then exteriorized and divided. A new pouch was constructed and subsequently placed in the abdomen. Further dissection of small adhesions was performed laparoscopically to ensure adequate reach. The new pouch was laid in the pelvis, to be guided transanally into the deep pelvis. A hand sewn anastomosis was then performed using interrupted stitches of 2-0 VicrylThe patient recovered uneventfully on the surgical ward and was discharged on postoperative day 3. The loop ileostomy was reversed at 8 weeks, after satisfactory endoscopic and radiologic assessments of pouch integrity.To our knowledge, we present the first description of the transanal approach as an alternative for difficult IPAA excision. It is possible that the transanal approach provides a less hostile field, for the surgeon to re-enter the pelvis, when compared with a purely transabdominal approach. Perhaps the benefits of minimally invasive surgery can still serve the patient. It is imperative that surgeons attempting this procedure have an extensive experience in IPAA surgery and taTME approach.Below is the link to the electronic supplementary material.Supplementary material 1 (MP4 370812 KB)


Diabetes mellitus (DM) is the leading cause of polyneuropathy in the Western world. Diabetic neuropathy (DNP) is the most common complication of diabetes and is of great clinical significance mainly due to the pain and the possibility of ulceration in the lower limbs. Early detection of neuropathy is essential in the medical management of this complication. Early unmyelinated C-fiber dysfunction is one of the typical findings of diabetic neuropathy and the first clinical manifestation of dysfunction indicating sudomotor eccrine gland impairment. In order to assess newly developed technology for the measurement of dermal electrochemical conductance (DEC), we analyzed the feasibility and effectiveness of DEC (quantitative expression of sudomotor reflex) as a screening test of DNP in primary health care centers. The study included 197 people  who underwent all the protocol tests and electromyography (EMG). On comparing DEC with EMG as the gold standard, the area under the receiver operating characteristic (ROC) curve  was 0.58 in the whole sample, AUC = 0.65 in the diabetes population and AUC = 0.72 in prediabetes, being irrelevant in subjects without glucose disturbances (AUC = 0.47). Conclusions: In usual clinical practice, DEC is feasible, with moderate sensitivity but high specificity. It is also easy to use and interpret and requires little training, thereby making it a good screening test in populations with diabetes and prediabetes. It may also be useful in screening general populations at risk of neuropathy. The prevalence of diabetes mellitus (DM) is very high in Spain, being approximately 14% according to oral glucose tolerance test (OGTT) results . Indeed,PN is defined as the presence of symptoms and/or signs of peripheral nerve dysfunction in people with DM, after ruling out other possible causes . The TorThe most common clinical presentation of PN is distal symmetric polyneuropathy (DSPN), being predominantly sensory in 80% of cases . Pain isIn the Rochester study including >64,000 patients, the prevalence of PN was between 66% and 59% for type1 DM and type 2 DM, respectively . The 3rdDSPN-related factors are age, DM duration, metabolic control, male gender, acute myocardial infarction, hyperlipidemia , smoking, and general cardiovascular risk factors ,15,16,18There are many confirmatory tests, including measurements of nerve conduction velocity (EMG) and biothesiometry or skin biopsy. However, the most commonly used are the measurement of altered sensations using a vibrating tuning fork with 128 Hz and/or pressure with Semmes-Weinstein 5:07 monofilament . MonofilAlthough electrophysiological measures are more objective and reproducible, they are limited in that they only detect dysfunction based on the presence of thicker and faster (myelinated) fibers and show their involvement later. Consequently, EMG is a specific, albeit very insensitive, test. Recently developed noninvasive techniques are more reproducible and reliable for the detection of early dysfunction of small fibers. One of these new techniques involves the measurement of dermal electrochemical conductance (DEC) or the sudomotor dysfunction index and has been evaluated by well-designed studies which support its use as a screening test ,19,20,21Ramachandran et al.  studied Although we will not discuss the best algorithm to screen diabetic neuropathy, certain tests have shown promising results in predicting future diabetes-related complications. Some of these tests include related factors such as obesity, macroangiopathy  or retinopathy ,26,27,28The hypothesis of our study was that the measurement of DEC is feasible, sensitive, and specific and more or equally effective to other techniques commonly used in the initial screening of diabetic neuropathy in primary care.To evaluate the feasibility, effectiveness and performance of a new technique which measures DEC (sudomotor reflex) in the screening of diabetic neuropathy in primary care.Determine the performance of DEC (quantitative assessment of sudomotor reflex) as a tool for the screening of diabetic neuropathy in primary care compared with the Semmes-Weinstein 5:07 MFT when an EMG is used to confirm the presence of diabetic PN in patients with prediabetes and type 2 diabetes.Determine the performance of DEC (quantitative assessment of sudomotor reflex) as a tool for screening diabetic neuropathy in primary care compared with the Semmes-Weinstein 5:07 MFT when the NDS is used to confirm the presence of diabetic PN in patients with prediabetes and type 2 diabetes.®, Impeto Medical, Paris, France) and EMG; with MFT 5.07 (10 g) Semmes-Weinstein; with Rydel-Seiffer 128 Hz tuning fork (vibrating sensibility); and the NDS score, in a consecutive sample of individuals attended in the primary care setting in the Vallès Occidental and Baix Camp counties , and their reference hospitals.The study design has been extensively described elsewhere . In brieWe performed a blind, prospective study comparing DEC (sudomotor reflex) , EMG, the Semmes-Weinstein 5:07 MFT (10 g), the sensitivity of a vibrating tuning fork 128 Hz, NDS score and DN4 score in a consecutive series of patients treated in primary care.The primary care teams of Terrassa-Sud and other partners belonging to the Mútua Terrassa reference hospital and those of the primary care teams of Reus (CAP Sant Pere) and the University Hospital Sant Joan de Reus reference hospital participated in the study.We consecutively included patients with type 2 DM over 40 years of age, with or without symptoms of neuropathy, attended in primary care. We also included the following 2 groups of patients matched by age and sex: one including patients with prediabetes ) and/or impaired glucose tolerance (IGT) determined by OGTT after 2-h 75 g oral glucose administration and another including patients without glucose alterations  (control group).Three main diagnostic categories  were defined using the WHO criteria based on 2-h postload glucose (<7.8 (140 mg/dL), 7.8–11.0 mmol/L (140–200 mg/dL)) and/or fasting plasma glucose  and >11.1 mmol/L (>200 mg/dL), respectively.The exclusion criteria were type 1 DM, upper or lower limb amputation , diagnosis of neuropathy not related to diabetes, neuropathy by entrapment, use of psychoactive substances, chronic alcoholism, malnutrition; treatment with beta-blockers, presence of terminal disease, or life expectancy <3 years. Pregnancy was ruled out in women (negative pregnancy test) and a history of gestational diabetes was also taken into account.The study period was from 1 January 2017 to 31 January 2019.It was estimated that the study should include a total of 160 participants in order to evaluate the validity and performance of a screening test showing a sensitivity of 82%, a precision of 9%, and a confidence interval of 95% (95% CI: 128–192), and considering a drop-out rate of 20%. The proportion of diabetes/prediabetes/normal glucose tolerance was 2:1:1.The contribution of patients by centers was 66% from the Mutua de Terrassa (minimum 106 cases) and 34% from Reus (minimum 54 cases), achieving a final sample size of 197 cases. This has been described previously elsewhere . In brieAfter verifying the inclusion criteria and receiving written informed consent to participate, the medical history of the patients was obtained and a physical examination was performed using the MFT, and the NDS score was given to screen for PN. The patients also underwent DEC quantification using the Sudoscan device. A score of 8 out of 8 with the Semmes-Weinstein 5:07 MFT (10 g) was considered as sensitive . An NDS Done at the reference hospital, where a neurologist blinded to previous test results, performed a neurographic test, including a sensory conduction study of the median, ulnar and sural nerves, and motor conduction study of the deep peroneal nerve. The variables studied included the amplitude of compound muscle action potential and distal latency of the motor nerves, and amplitude and distal latency of sensory nerves. DEC determination and the other neuropathy screening and electrophysiological tests took no longer than 1 month.The results of the previous visit were recorded and the patients were informed of the results and the diagnosis.2 test was used to analyze qualitative variables and the Student’s t test was performed for quantitative variables. Logistic regression was used to identify predictors of diabetic neuropathy. Dependent variables (response) included the presence of diabetic neuropathy diagnosed by EMG or the NDS questionnaire. The performance of DEC was compared with the MFT as screening tests of PN. To determine the validity and reliability of DEC, the sensitivity, specificity, the PPV and NPV, and the positive and negative likelihood ratios were calculated. A receiver operating characteristic (ROC) curve was used and the area under the curve was calculated. A p < 0.05 was considered as statistically significant. The analyses were performed with the statistical packages STATA/SE 12.0  and R for Windows .The χwww.idiapjordigol.org) approved the protocol  and each participant signed written informed consent.The research Ethics Committee board at the Jordi Gol Research Institute (Barcelona)  curve (AUC) of 0.58. The high specificity found indicates that DEC is a reliable test for the detection of individuals without PN while the low sensitivity suggests that this test is not very useful for the detection of disease among people with PN. On comparing DEC with other tests such as the MFT, NDS or DN4 questionnaires, the sensitivity, specificity, PPV and NPV were similar, in the whole population and in individuals without DM and in those with prediabetes . Painful neuropathy (DN4 >4 points) was observed in a total of 12 patients: 11 in the diabetes group (11%) and 1 in the prediabetics (0.02%) and none in the group with normal tolerance.In the whole sample, the ROC curve comparing DEC with MTF (AUC = 0.65) showed greater effectiveness than that comparing DEC vs. EMG (AUC = 0.54). On the other hand, in the population with type 2 diabetes, the comparison of DEC with EMG showed the greatest significance (AUC = 0.66). Although the results are not very high, DEC may be a good screening test for populations with PN compared to the gold standard (EMG). It was of note that in the screening of prediabetic subjects the AUC was 0.72, which is reasonable for a screening test. As mentioned in the Introduction, state-of-the-art methods demonstrate a high variability in PN diagnostic methods, being directly related to the variability in prevalence rates. For example, with the use of EMG, Puig and Portillo obtained prevalences of 75 and 86%, respectively ,25. HoweOne important difference of our study is that we also evaluated a group with prediabetes. With the addition of this group, the definitive results differ from the preliminary results, with the NDS questionnaire showing the best AUC compared to MFT before the addition of prediabetes. There are few studies in this regard. However, some have suggested the utility of DEC for detecting diabetes in subjects with glucose intolerance, being even better than basal glycemia. With the use of DEC in 212 subjects, Ramachandran  diagnoseIn a study evaluating 47 patients with type 2 diabetes and 16 controls, Kneger et al.  reportedBins-Hall et al.  studied In a Chinese cohort, Sudoscan™ was a useful method to detect PN in a screening of 394 asymptomatic persons with diabetes .In a systematic review and meta-analysis on MFT, Wang et al.  reportedIt is of note that in present study we prioritized the NDS and DN4 scores; the first provides clinical data (questionnaire) and exploration data  while the second is an abbreviation of the first, and provides a greater amount of clinical data. The main limitation of the present study is accurate diagnosis of diabetic neuropathy since some studies have shown that some cases of diabetic neuropathy present no alterations in the EMG. Indeed, both EMG and MFT examine large nerve fibers. However, since they are the usual tests available, they represent a limitation of the study. Therefore, several considerations should be taken into account. First, the EMG test is more specific, albeit not very sensitive, showing positive results in advanced stages of PN. The fingerboard and the NDS questionnaire are commonly used for the diagnosis of diabetic neuropathy, probably because the NDS is carried out before EMG and is actually often used to avoid the need for EMG.Another inherent limitation of this study may be the low number of participants, although this number fulfilled that obtained in the calculation of the sample size. Therefore, both the EMG and the NDS score, which mainly assess the dysfunction of myelinated fibers, provide a good profile for diagnostic confirmation. On the contrary, both the DEC and the MFT are able to diagnose and stage diabetic neuropathy earlier than the previous 2 tests by the detection of unmyelinated fiber dysfunction. Nonetheless, another limitation is that MFT is a good test for the prediction of foot ulcers but is certainly insensitive for the detection of early neuropathy. Therefore, for purposes of simplification and taking into account the possible limitations, we compared the effectiveness of the measurement of DEC and the use of MFT as diagnostic tools in primary care according to whether the true diagnosis is achieved by the EMG or the score of the NDS questionnaire.Although other tests are available to detect early neuropathy, such as the Utah Early Neuropathy Scale (UENS) and the Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (QOL-DN) scale, these questionnaires are not practical in the clinical scenario. However, the latter test is a good tool to detect neuropathy unawareness in patients with diabetes . Indeed,In regard to technical aspects, the use of DEC in clinical examinations has a few limitations as described in previous studies . However, we do not believe that these aspects apply to the present study since they were exclusion criteria and were not affected by age, sex or previous physical exercise or changes in temperature or acute alterations in blood pressure. According to these studies, the results of DEC do not depend on the rate of perspiration , having One of the strengths of this study was that DEC and physical examinations were performed by only a few investigators in order to avoid biases in sample selection or erroneous categorization of the diagnosis of PN. This is one of the first studies to compare DEC measurement with the gold standard (EMG). Other studies, even those by the manufacturer, are based on other devices or clinical questionnaires, thereby making our study innovative. Considering the limitation of resources in primary care and the health care system as a whole, we are currently performing a cost-effectiveness analysis of the use of DEC in the primary care setting. In conclusion, in the usual clinical practice, DEC is feasible, with a moderate sensitivity but a high specificity. It is also easy to use and interpret and requires little training, thereby making it a good screening test in populations with diabetes and prediabetes. It may also be useful in screening general populations at risk of neuropathy. Nonetheless, the results of cost-effectiveness analyses must first be analyzed before recommending the implementation of DEC in primary care in Catalonia.


Hibiscus splendens and Solanum echinatum, and contrasted with exudates of Sorghum bicolor, a plant widely demonstrated to exhibit BNI capacity. Root exudates were collected from plants at two, four, and six weeks after transplanting to solution culture. Root exudates contained three types of organic acids (OAs), oxalic, citric and succinic acids, regardless of the species. However, the two Australian natives species released larger amount of OAs in earlier development stages than S. bicolor. The total quantity of these OAs released per unit root dry mass was also seven-ten times greater for Australian native plant species compared to S. bicolor. The root exudates significantly inhibited nitrification activity over six weeks’ growth in a potential nitrification assay, with S. echinatum (ca. 81% inhibition) > S. bicolor (ca. 80% inhibition) > H. splendens (ca. 78% inhibition). The narrow range of BNI capacity in the study plants limited the determination of a relationship between OAs and BNI; however, a lack of correlation between individual OAs and inhibition of nitrification suggests OAs may not directly contribute to BNI. These results indicate that Australian native species generate a strongly N conserving environment within the rhizosphere up to six weeks after germination, establishing a competitive advantage in severely N limited environments.Australian native species grow competitively in nutrient limited environments, particularly in nitrogen (N) limited soils; however, the mechanism that enables this is poorly understood. Biological nitrification inhibition (BNI), which is the release of root exudates into the plant rhizosphere to inhibit the nitrification process, is a hypothesized adaptive mechanism for maximizing N uptake. To date, few studies have investigated the temporal pattern and components of root exudates by Australian native plant species for BNI. This study examined root exudates from two Australian native species,  Andropogon gayanus and Bromus tectorum  and phosphorous (P) are generally the most commonly limiting nutrients in terrestrial ecosystems, depending on ecosystem development and input processes . In parttectorum . Weed sps growth .With the exception of some native African grasses , to dateSorghum bicolor root exudates including, 3-(4 hydroxyphenyl) propionate (MHPP), sakuranetin, and sorgoleone have been identified as active compounds for BNI  root exudates of selected Australian native plant species inhibit the rate of nitrification; and (2) the BNI activity of root exudates is correlated with total concentrations, or specific types, of anionic organic acids.Hibiscus splendens and Solanum echinatum, were selected because of their adaptation to slightly acidic, light textured soils with low soil N content, as determined by the CSIRO soil map . S. bicolor was examined as a positive control due to documented BNI by its root exudates .Four germinated seedlings of each species were transplanted to a solution culture system. The solution nutrient content was adjusted to respective nutrient concentrations similar to those typically found in soils , with a H of 5.6 . The nut4Cl  analyses at the Queensland Department of Science, Information Technology and Innovation . The two UHPLC methods utilized were based on parameters detailed in w 5.8 and nitrifying potential ca. 0.63 mg NO−3 L−1 h−1, collected from the Darling Downs . Six grams of field-moist soil with 40 mL of NH+4 substrate solution, comprised of 3.8 mM (NH4)2SO4 made with 1 mM phosphate buffer solution at pH 7.2, was placed into a 100 mL flask. Concentrated exudate samples in 200 µL DMSO were re-suspended in 1 mL deionised (DI) water and added to the soil-slurry. A negative control was included, containing 200 µL DMSO made up to 1 mL with DI water to eliminate the effects of DMSO from the evaluation. Flasks were sealed with Parafilm® with three pin holes per flask to allow gas exchange but prevent moisture loss. All flasks were placed on an orbital shaker incubator at 180 rpm and 25 °C. Samples of soil slurry were collected at times between 0 and 24 h, then centrifuged (3000 rpm for 10 min) to separate solid and liquid phases. The supernatant was filtered to 0.45 µm and stored at −20 °C until further analysis. The concentrations of NO−2 in soil slurry supernatants were determined colorimetrically using sulphanilamide and N-(1-naphthyl)-ethylene-diamine dihydrochloride  was calculated based on the difference between initial (0 h) and subsequent (22 h) measured concentrations of NO3-N. Negative values for nitrification potential were removed from the analysis and figures. The end time of incubation was determined by the concentration of NO3-N in control as the method assumes a linear increase of NO−3. In this study, the linear increase of NO3-N was observed until 22 h (data not shown); hence, 22 h data were treated as the end-point samples. Nitrification measurement at time zero allowed accurate interpretation of NO3-N production by removing potential discrepancies associated with varying concentrations of indigenous NO3-N in the test soil. The nitrification rates were calculated as the average concentration (mg N L−1), minus the initial concentration (0 h), divided by the number of hours in incubation. The extent of BNI was estimated as the percent reduction in nitrification rate of soil incubated with exudates, compared to nitrification rates in untreated soil (control). Organic acid production by individual plants (mg plant−1 day−1) was determined using concentration data (mg L−1) multiplied with the concentrated sample volume (0.02 L), and then divided by the number of plants per pot. Two-way analysis of variance (ANOVA) was used to determine the effects of plant species and age on NO3-N values in soil incubations, both with root exudates and without (control included). Post-hoc Tukey pairwise comparison was used to determine grouping of significant effects at p = 0.05. To determine influencing factors without the masking effect of control values, both production of NO3-N and BNI capacity from soil incubated with root exudates (control excluded) were analysed by two-way ANOVA with post-hoc Tukey analysis for the effects of plant species and age.Nitrification potential  in soil solution, relative to the uninhibited soil , than S. echinatum (ca. 0.0285 mg NO3-N L−1 h−1). The positive control, S. bicolor, exhibited a nitrification rate intermediate between the two Australian native species (0.0296 mg NO3-N L−1 h−1) (The exudates of both ted soil . Over siL−1 h−1) .S. bicolor demonstrated intermediate BNI activity (80%), with S. echinatum exudates exhibiting slightly stronger inhibition (81%) than that of H. splendens (78%) (p > 0.05). All three plant species demonstrated effective BNI at age two, four, and six weeks, with exudates of S. echinatum exhibiting the greatest effect on soil nitrification over six weeks.The BNI activity, indicated as the percent reduction in nitrification rates in soil treated with root exudates, varied with plant species and age but not significantly . Over sins (78%) . Whilst S. bicolor exuded the greatest quantity of the OAs examined, yielding a total of 3.7% more OA than S. echinatum and 26.2% more than H. splendens  and S. echinatum (79 µg L−1) were similar, H. splendens released much less at 65 µg L−1 , which conversely suggests little correlation of OAs to BNI capacity . Investigation of root exudate OA content across a suite of plants exhibiting a range of BNI capacities is required to conclusively determine the relationship of OA to BNI. Analysis of the interrelationship between individual OA concentrations and NH4-N oxidation inhibition . Copper is required for the activation of the ammonia-oxidising enzymatic pathway , and welThe release of OAs is likely a general response of establishing plants to enhance nutrient availability , which mUnderstanding the role of BNI in establishment and persistence of native species is important when considering natural landscapes, ecological succession, and revegetation programs. Nitrogen availability is a major driver of succession in vegetation states , with imH. splendens and S. echinatum. Organic acids, while present, may not have a direct and/or individual role in BNI function in root exudates of Australian natives. The previously unexamined class of plants provides a new category of plant species for investigation of the major active components and function of BNI. The present study identifies an active physiological process in native plants, which is an important consideration for native ecology.This study has provided the first evidence for a high degree of BNI activity in root exudates of Australian native plant species 10.7717/peerj.4960/supp-1Supplemental Information 12-N and NOx to determine the level of nitrification activity. Measurements were taken over 0–24 h.Recorded measurements of NOClick here for additional data file.10.7717/peerj.4960/supp-2Supplemental Information 2High liquid performance chromatography, using ultra-violet was used to determine the presence and concentration of organic acids in a sub-sample of root exudates. Raw results are presented in a table.Click here for additional data file.10.7717/peerj.4960/supp-3Table S1Biomass data after 42 days of growth in nutrient solution culture. Table shows the mean wet and dry mass, and the mean moisture content, of each plant species.Click here for additional data file.10.7717/peerj.4960/supp-4Figure S1Growth of the major stem of plant over 42 days. Measurements taken from the main stem at the highest node to the beginning of the root system.Click here for additional data file.10.7717/peerj.4960/supp-5Figure S2Growth of the root system over 42 days. Measurements taken from the tip of the longest root to the initiation of the root system from the stem. Data here is not indicative of total growth, refer to Click here for additional data file.


Rhizomucor miehei was expressed in Yarrowia lipolytica, which then showed a galactosidase activity of 121.6 U/mL. Under the effects of the recombinant α-galactosidase, most of the raffinose-family oligosaccharides in soy molasses were hydrolyzed into sucrose. Then the soy molasses hydrolysate with high sucrose content  was supplemented into the medium, with an isomaltulose production of 209.4 g/L, and the yield of 0.95 g/g. Finally, by virtue of the bioremoval process using Pichia stipitis, sugar byproducts in broth were transformed into ethanol at the end of fermentation, thus resulting in high isomaltulose purity (97.8%). The bioprocess employed in this study provides a novel strategy for low-cost and efficient isomaltulose production from soybean molasses.Isomaltulose is mainly produced from sucrose by microbial fermentation, when the utilization of sucrose contributes a high production cost. To achieve a low-cost isomaltulose production, soy molasses was introduced as an alternative substrate. Firstly, α-galactosidase gene from  Isomaltulose is a kind of structural isomer of sucrose and natural reducing disaccharide. Although isomaltulose shares similar physical and organoleptic properties with sucrose, it has been approved as a safer sucrose substitute with advantages including slower release and absorption, higher stability, lower plasma glucose and insulin concentrations ,2,3. In Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Yarrowia lipolytica, have been employed as expression hosts. Using the engineered strains, increased SIase activity and maintained security were achieved [Y. lipolytica strain, in which a bacteria-derived SIase gene was expressed extracellularly, an isomaltulose production of 572.1 g/L from sucrose was achieved and the yield reached 0.96 (g/g), suggesting a huge potential for industrial isomaltulose production [Since chemical synthesis of isomaltulose is accompanied with some byproducts that are difficult to remove, bacterial fermentation became the primary technology adopted in industrial isomaltulose synthesis ,8. The bachieved ,11,12,13Bacillus subtilis.Another limiting factor for isomaltulose production is the relatively expensive price of the sucrose substrate. The cost of sucrose takes up more than 50% of the total cost. Therefore, introducing inexpensive feedstocks instead of pure sucrose as alternative substrate was economically feasible to lower the cost ,13,14. Cw/w), containing mainly stachyose, sucrose, raffinose, glucose, xylose, galactose, and fructose. Due to the high carbohydrate content and the relatively low cost, soy molasses have been of great interest among researchers and biorefinery industrialists to produce value-added chemicals. However, the current soy molasses-derived fermentations have limited efficiency, as most have focused on the utilization of the single constituents. Due to the high content of indigestible raffinose-family oligosaccharides (RFOs), referring to stachyose and raffinose, it was difficult for soy molasses to be completely degraded. The RFOs of soy molasses could not be converted into a usable product, and thus was discharged as waste from the process [Soy molasses is a low-value byproduct obtained during the extraction of soybean proteins and was generated largely with the increasing demand of high-quality soy protein . Sugar c process ,17,18,19 process ,19.To increase the utilization efficiency of soy molasses, fermentation and complex enzymatic methods have been adopted to remove the RFOs and convert them into digestible monosaccharides. Notably, in particular, the single enzyme α-galactosidase can hydrolyze RFOs into sucrose and galactose . α-galacY. lipolytica strain. Subsequently, an ethanol producing bioremoval process was employed to remove sugar byproducts and improve isomaltulose purity. These processes presented a new potential choice for low-cost isomaltulose synthesis in industrial production.In this study, we sought to achieve efficient isomaltulose production from soy molasses. A heterologously expressed α-galactosidase was introduced to digest soy molasses and increase sucrose content. Then transformed and native sucrose in soy molasses was served as substrate to produce isomaltulose using an engineered -Y. lipolytica ura was employed as a host to express α-galactosidase. Recombinant strain S47 used for isomaltulose production has been described previously [Pichia stipitis, Saccharomyces cerevisiae, Hanseniaspora uvarum, and Kluyveromyces marxianu, were preserved in the lab. The α-galactosidase gene used in this study was from Rhizomucor miehei (accession number: JF340459) and named as RMgase. RMgase gene manipulation was conducted in strain E. coli DH5α which was cultured in the Luria–Bertani (LB) medium. LB medium with the final concentration of 30.0 μg/mL kanamycin or 100.0 μg/mL ampicillin was prepared if necessary. YPD medium  was used for culturing yeast strains. YNB plate 2SO4, 25.0 g/L agar) was used for yeast transformant selection [2SO4, 6.0 g/L yeast extract, 2.0 g/L KH2PO4, 3.0 g/L K2HPO4, and 0.1 g/L MgSO4·7H2O with a pH of 6.0 [Uracil mutant strain eviously . Four etelection . GPPB meH of 6.0 .Y. lipolytica) signal peptide at its 5′ end was synthesized after codon redesign and optimization . The synthesized RMgase gene was then inserted into expression vector pINA1312 to obtain the reconstructed plasmid pINA1312-RMgase. The recombinant plasmid pINA1312-RMgase was linearized with NotI. Linearized DNA vector containing RMgase gene was transformed into Y. lipolytica URA- cells by the LiAc method [Y. lipolytica URA- cells. The yeast cells were spread on the above-mentioned YNB plate to select the positive transformants and then cultured at 28 °C for 3 days. Genomics DNA from these transformants owing the relative higher α-galactosidase activity were extracted and used to amplify the RMgase gene with previous primers. Transformants were cultivated in YPD liquid medium at 30 °C for 20 h and transferred into GPPB medium for 3 days. Among a total 216 these transformants, G82 had the highest extracellular α-galactosidase activity of 121.6 U/mL.The construction of the expression vector pINA1312 containing RMgase gene was executed according to the literature description [c method . As a co2CO3 and filtered through a 0.22-μm membrane for high-performance liquid chromatography (HPLC) analysis. An Agilent 1200 system  with NH2 column  was employed to detect the carbohydrates content. The carbohydrate concentration was calculated according to peak areas and retention time. The amount of enzyme releasing 1 μM p-nitrophenol per min at 45 °C and in buffer of pH 4.5 was defined as one unit of α-galactosidase activity (U). Furthermore, the α-galactosidase activity based on other three different substrates, including stachyose, raffinose and melibiose, were assessed. Three independent assays were carried out and the average values were calculated.Detection of α-galactosidase activity detection was evaluated using pNPG as substrate . The reag, and the α-galactosidase activity in the supernatant was detected (see below). The RMgase was purified by DEAE–Sepharose Fast Flow chromatography . The effect of pH on purified enzyme activity was assessed with the enzyme equilibrated in different reaction citrate-Na2HPO4 buffer (3.0–8.0) for 2 h min at 45 °C. After the α-galactosidase enzyme solution (4 °C) was subjected to different pH values (3.0–8.0) for 2 h, the pH stability was evaluated by detecting residual activity. To further investigate the influence of temperature on activity, α-galactosidase activity detection was assayed at temperatures ranging from values (40–70 °C) with pH 4.5. Enzyme thermo stability was performed by estimating remaining activity after a 2 h incubation at pH 4.5 with different temperatures values (40–70 °C). Means and standard errors from three replicates per point are shown.The culture was centrifuged at 5000× w/w) stachyose, 13.42% (w/w) sucrose, 3.32% (w/w) glucose, 1.73% (w/w) xylose, 0.78% (w/w) galactose, 2.81% (w/w) fructose. The crude fermentation broth of recombinant G82 with the activity of 121.6 U/mL, was used for soy molasses digestion. The pretreated soy molasses was hydrolyzed by α-galactosidase for 4 h at 45 °C and pH 4.5. The soy molasses hydrolysate (SMH) was collected and their composition was analyzed by HPLC. Furthermore, different dosages of α-galactosidase based on the amount of soy molasses with a range from 0 U/g to 20 U/g was added into the soy molasses to hydrolyze stachyose and raffinose completely. Sugar content was detected by HPLC analysis.The soy molasses was provided from a local soybean oil factory. The soy molasses was pretreated using the chemical method described in the previous study . The soyY. lipolytica S47 were inoculated in GPPB broth for 36 h. Then, SMH ranging from 400 g/L to 800 g/L were respectively added into the flask for another 48 h. The products were detected, and three replicates were used for each experiment.The optimized two-stage bioprocess, including the first growing stage and the second producing stage, was proved to be an effective fermentation strategy for food-grade isomaltulose because of high production, elevated yield, and reduced byproduct . A two-sTo validate the optimized conditions obtained above, a large-scale isomaltulose production using a two-stage bioprocess was conducted in a 10-L bioreactor. Following this, 200 mL strain S47 culture was prepared and inoculated into a 10-L Biostat B fermenter  with a 6-L GPPB fermentation medium. The fermentation for isomaltulose production was conducted with agitation speed of 300 rpm, aeration rate of 5 L/min at 30 °C and pH 6.5. After the fermentation continued for 32 h, 700 g/L SMH broth was supplemented into the bioreactor to ferment another 16 h. The concentration was of the SMH broth, not of the sucrose. At 48 h, another 300 g/L SMH broth was supplemented into the bioreactor. For the analysis of SIase activity, sugar concentration, biomass and isomaltulose production during the fermentation, three parallel samples were taken from the fermenter regularly and were detected as described in a previous study .P. stipitis, S. cerevisiae, H. uvarum, and K. marxianu, were used to check their ability to eliminate the residual galactose and xylose as Li description with some changes [Y. lipolytica was used as a control. Briefly, the single colony from four yeast strains were respectively inoculated into YPD broth at 30 °C overnight as cell culture preparation. The ethanol-producing processes were carried out parallelly in 1-L fermenters. Following this, 600 mL of the final isomaltulose fermentation broth obtained in To further reduce the carbohydrate byproducts and improve the isomaltulose purity, four ethanol-producing yeast strains, including  changes . Y. lipoY. lipolytica strain G82 had the highest extracellular α-galactosidase activity of 121.6 U/mL, which was higher than that of Penicillium sp. (6.03 U/mL), Rhizopus sp. (1.69 U/mL), Gibberilla sp. (1.42 U/mL), and Absidia corymbifera (18.7 U/mL) [Escherichia coli carrying the same α-galactosidase gene (11.03 U/mL) [In this study, α-galactosidase was expected to release free sucrose from RFOs in soy molasses. Recombinant .7 U/mL) ,28,29,30The effects of pH and temperature on enzyme activity and stability were evaluated according to former literature ,31. The Talaromyces emersonii expressed in the yeast Pichia pastoris lost most activity when the pH was beyond 5.5 [Many α-galactosidases had poor pH stability. For example, the α-galactosidase from the yond 5.5 . Howeveryond 5.5 ,34.w/w) and 1.64-fold than those in the undigested molasses.To increase sucrose content in soy molasses, the recombinant α-galactosidase was used to hydrolyze the RFOs. A temperature of 45 °C and pH of 4.5 were selected as the appropriate conditions for the process. Under the conditions, the recombinant α-galactosidase exhibited good activity and stability. The influences of different α-galactosidase adding dosage on soy molasses composition were analyzed. Accompanied by the α-galactosidase adding dosage rising, RFOs in the soy molasses both had a remarkable reduction. The raffinose and stachyose were almost converted into sucrose and galactose when the adding dosage was beyond 15 U/g d. The fiThe RFOs were hardly directly utilized by microbes, resulting in a low feedstock conversion rate ,35. NotaY. lipolytica S47 lacks invertase activity; therefore, SIase-catalyzing isomerization was the only pathway consuming sucrose in SMH. Strain S47 utilized glucose and fructose to maintain viability, which existed in SMH or was generated during SIase-catalyzing isomerization. Different SMH supplementation concentrations were investigated to estimate the optimal fermentation condition. The isomaltulose production had a continuous enhancement with the increase of SMH. The isomaltulose yield remained stable with a concentration range from 400 to 700 g/L and began to dramatically decrease at 800 g/L with a yield of 0.86 (g/g)  . Since a86 (g/g) . In the 86 (g/g) . In the a strain . HoweverY. lipolytica cells. Corresponding to the sucrose content in SMH, the isomaltulose production reached 209.4 g/L. This value was much higher than that of engineered L. lactis (36 g/L) and engineered S. cerevisiae (< 4 g/L) using pure sucrose as a substrate [B. subtilis, with a yield of 0.924 (g/g). Due to the higher native sucrose content in cane molasses, the production of 221.6 g/L was slightly higher than that in this study [As mentioned above, sucrose in SMH was isomerized into isomaltulose during the bioprocess. The yield of 0.95 (g/g) was very close to that of 0.96 (g/g) using pure sucrose as a substrate in our previous study . This yiubstrate ,12. Caneis study . Howeveris study . Thus, sActually, constructing a single strain with both α-galactosidase and SIase activities can simplify the processes. Undesirably, by tandemly expressing the SIase gene and galactosidase gene, the activities of both two recombinant enzymes were fairly low (data not shown). We speculated that this may be due to use of the same hp4d promoter for the expression of two genes. Thus, finding a new suitable promoter is the key point. In this study, a different strain G82 was constructed to gain high galactosidase activity.S. cerevisiae, K. marxianus and P. stipitis; xylose can only be removed by P. stipitis; no loss of trehalulose and isomaltulose was observed after treatments by all the four strains. Therefore, P. stipitis was identified as the optimal strain for bioremoval. After a distillation process, only isomaltulose (209.4 g/L) and trehalulose (4.6 g/L) remained in the broth. On the whole, by combining enzymatic hydrolysis and fermentation, 1000 g soy molasses was converted into 209.4 g isomaltulose and 32.9 g ethanol. Based on the much higher value of isomaltulose than ethanol, the ethanol production occupied a small percentage of the total output. However, ethanol production to remove other sugars played an important role for efficient extraction of isomaltulose from the broth. As solubilities of the monosaccharides were much larger than that of isomaltulose, the residual monosaccharides of high concentrations can weaken the extraction of isomaltulose, discarding large part of the product and reducing economic value.Apart from target product isomaltulose, galactose, xylose, and trehalulose remained in the final broth, with the concentration of 96.1 g/L, 17.3g/L and 4.6 g/L, respectively. To eliminate the undesirable sugars and avoid introducing new metabolites, four ethanol-producing yeast strains were employed to perform the bioremoval process. As shown in S. cerevisiae, then a higher trehalose purity (99.5%) was obtained [S. cerevisiae cannot utilize any kinds of undesirable sugars remaining in the final isomaltulose fermentation broth. Thus, non-traditional ethanol-producing yeast P. stipitis was screened to perform the bioremoval. After the treatments by P. stipitis, isomaltulose accounted for 97.8% of the total sugars, much higher than the proportions of generally below 90% in other isomaltulose productions. The results demonstrate that the bioremoval process adopted in this study can help achieve high-purity isomaltulose production. Additionally, the use of P. stipitis can achieve removing more kinds of specific undesirable sugars and broadened the application of ethanol-converting bioremoval method.For functional sugar manufacturing, additional sugar byproducts were always followed with the target product. Current sugar separation methods were based on the chromatographic technique, which is not only laborious and time-consuming, but also very expensive. The use of yeast cells to remove specific sugars can be a promising alternative method. According to the discrepancy of utilizing ability for different sugars, undesirable sugar products can be consumed by the cells, while target product remained. In trehalose synthesis, the remaining maltose substrate and byproduct glucose were both converted into ethanol by obtained . Howeverw/w) by α-galactosidase hydrolysis. SMH was used as the substrate, with isomaltulose production of 209.4 g/L, and a yield of 0.95 (g/g). Finally, by virtue of the bioremoval process using P. stipitis, isomaltulose accounted for 97.8% of the total sugars. Therefore, this bioprocess is expected to provide a basis for further development of efficient and low-cost isomaltulose production. Construction of engineered strains performing the three steps simultaneously would simply the bioprocess.In this study, a three-step linkage strategy for isomaltulose production from soybean molasses was established. The sucrose content in the soy molasses was increased to 22.04% (