In March 2020, South Africa implemented strict nonpharmaceutical interventions (NPIs) to contain the spread of COVID-19. Over the subsequent 5 months, NPI policies were eased in stages according to a national strategy. COVID-19 spread throughout the country heterogeneously; the disease reached rural areas by July and case numbers peaked from July to August. A second COVID-19 wave began in late 2020. Data on the impact of NPI policies on social and economic well-being and access to health care are limited.We aimed to determine how rural residents in three South African provinces changed their behaviors during the first COVID-19 epidemic wave.The South African Population Research Infrastructure Network nodes in the Mpumalanga (Agincourt), KwaZulu-Natal,  and Limpopo  provinces conducted up to 14 rounds of longitudinal telephone surveys among randomly sampled households from rural and periurban surveillance populations every 2-3 weeks. Interviews included questions on the following topics: COVID-19–related knowledge and behaviors, the health and economic impacts of NPIs, and mental health. We analyzed how responses varied based on NPI stringency and household sociodemographics.In total, 5120 households completed 23,095 interviews between April and December 2020. Respondents’ self-reported satisfaction with their COVID-19–related knowledge and face mask use rapidly rose to 85% and 95%, respectively, by August. As selected NPIs were eased, the amount of travel increased, economic losses were reduced, and the prevalence of anxiety and depression symptoms fell. When the number of COVID-19 cases spiked at one node in July, the amount of travel dropped rapidly and the rate of missed daily medications doubled. Households where more adults received government-funded old-age pensions reported concerns about economic matters and medication access less often.South Africans complied with stringent, COVID-19–related NPIs despite the threat of substantial social, economic, and health repercussions. Government-supported social welfare programs appeared to buffer interruptions in income and health care access during local outbreaks. Epidemic control policies must be balanced against the broader well-being of people in resource-limited settings and designed with parallel support systems when such policies threaten peoples’ income and access to basic services. Since the emergence of COVID-19 in humans in late 2019, the epidemic has spread to every country in the world, resulting in direct mortality and morbidity . ShortlyThe lockdown was intended to (1) reduce COVID-19 transmission through strictly restricting physical interactions; (2) prevent rapid epidemic growth and allow health care providers to prepare for a subsequent rise in the demand for care; (3) promote widespread educational campaigns to reduce COVID-19 transmission; and (4) initiate an ambitious, country-wide, community-based COVID-19 screening and testing program [Although the cumulative number of COVID-19 cases slowly grew during April 2020, the disease incidence curve rapidly rose from May onward, peaking between June and August. By September 1, 2020, South Africa had reported over 625,000 confirmed COVID-19 cases and over 14,000 deaths ; the COVThe relaxation of lockdown regulations, which occurred even as the epidemic grew, reflected competing health and economic vulnerabilities and priorities as well as sustained popular pressures -15. TherRobust data are essential for evaluating the hypotheses that lockdowns cause substantial harm or are futile and for targeting locations that are the most in need of resources. The impact of NPIs has been evaluated in various low- and middle-income countries, generating evidence of early reductions in income and food security and the rapid, substantial uptake of protective behaviors -22.imilindo  [Although South Africa has effective national health care surveillance systems, there is limited capacity for monitoring the social and behavioral effects of NPIs on the COVID-19 epidemic at a local level. NPIs, such as those implemented in South Africa, might be expected to generate differing risks and benefits across socioeconomic settings. For example, there was unease in rural areas regarding water access for hand hygiene and d rooms) , while pd rooms) . The numTo date, most evaluations of the impact of COVID-19 in South Africa have been limited to web-based or urban settings -27. StudThe NIDS-CRAM has substantial strengths in terms of its national reach and wide-ranging topic coverage. However, it only provides sporadic snapshots of the epidemic. We therefore used an existing research infrastructure in three South African provinces to evaluate how health, social, and economic behaviors continuously changed between April and December 2020. We used a high-frequency survey of a panel of households for which substantial, pre-epidemic data were already available. Our first hypothesis was that behaviors would change as regulations and the national epidemic changed. Our second hypothesis was that these changes would vary based on socioeconomic characteristics  and households’ needs and resources.The South African Population Research Infrastructure Network (SAPRIN)—an initiative that is hosted by the South African Medical Research Council and receives long-term from the National Department of Science and Innovation—integrates three Health and Demographic Surveillance System (HDSS) nodes for population and health surveillance: (1) the Medical Research Council (MRC)/Wits Rural Public Health and Health Transitions Research Unit (Agincourt) in Ehlanzeni district, Mpumalanga ; (2) theIn March 2020, SAPRIN initiated plans for each HDSS node to implement a high-intensity, longitudinal telephonic survey that covered at least 750 randomly selected households in each province, by using telephone numbers extracted from each node’s most recent census. This sample was selected to obtain estimates of survey- and wave-specific proportions with a precision of no less than 4 percentage points, under the assumption of an 80% response rate based on past SAPRIN surveys. Every 2-3 weeks, a central call center at each node contacted households and asked a primary respondent to answer questions on behalf of the household. Primary respondents had to be resident adult members of the household (aged ≥18 years). To combat survey fatigue, from mid-September onward or after the seventh survey wave (whichever was earlier), one-third of the cohort was rotated out at each subsequent wave, and a new random sample that included the same number of households was rotated in for 4-6 survey waves. Details for each node are shown in Figure S2 in The questionnaire included both household-level and individual-specific questions; the latter could be directly answered by other household members if they were present. Otherwise, the primary respondent served as a proxy. The questionnaire included COVID-19 symptom screening; individuals who met the Department of Health’s COVID-19 symptom criteria were referred for further investigation, possible testing, and care. Data were captured on laptop computers by call center interviewers who used electronic data capture software, including automated skip patterns and validation checks. Telephone calls continued from April to December 2020 with continuous quality monitoring. The survey implementation process at one node (AHRI) is described in detail elsewhere . The stuOur outcomes for this study were based on answers to questions related to COVID-19 and NPIs in three key domains: (1) COVID-19–related knowledge and behavior; (2) the health and economic impacts of NPIs; and (3) mental health. For behavior, the primary respondent was asked to rate their perceived knowledge about COVID-19 on a 5-point scale; we classified respondents as (1) those who self-reported that they did not have enough knowledge or (2) those who reported that they had enough or more than enough knowledge. Respondents were then asked about household behavior changes that they made in response to the COVID-19 epidemic. They were asked whether any resident household member had left the house in the past 7 days and whether any nonhousehold members had visited the house during the preceding day; we classified respondents as either (1) those who reported any number of household or nonhousehold members or (2) those who reported “none” for each question. Respondents were also asked if household members had, over the past 7 days, avoided crowded areas or social events, travelled , or used face masks when going out.For health and economic impacts, primary respondents were asked about household members’ ability to (1) access all needed daily medications, (2) access needed health care, and (3) earn money. Finally, for mental health, we asked primary respondents to answer the Generalized Anxiety Disorder 2-item (GAD-2) and Patient Health Questionnaire 2-item (PHQ-2) scales. GAD-2 and PHQ-2 scores of ≥3 were considered positive, as per the standard, South African–validated cutoffs . The CroWe linked data from the high-intensity SAPRIN survey to the following routine individual and household sociodemographic data, which were collected from households in 2019: the number of children, working-aged adults, and pension-aged adults; the maximum education level attained; node-specific asset index quintiles; levels of employment; and the receipt of unconditional social grants. South African noncontributory pensions are available to all citizens, permanent residents, or documented refugees aged >60 years, although pension applications require proof of status.In this study, we included anyone who was interviewed in 2020. First, we described survey response rates at each node and time period as well as key, pre–COVID-19 epidemic household characteristics. Second, we described changes in each of the 11 measures across the three domains  based on node and month of data collection. Third, we used multivariable regression models to assess independent predictors of our outcomes of interest via complete case analysis. For each outcome, we fitted a Poisson model with household-level random effects and robust SEs to calculate prevalence ratios. All models included variables for node, interview round number, month of the interview, and household characteristics. Data analysis was conducted with Stata version 15.1 (StataCorp LLC) and R version 4.0.2 (The R Foundation) . ResultsAll households previously provided consent to be contacted by phone and each primary respondent provided recorded, verbal consent. Households were not directly compensated for study participation; however, one node annually provided a shopping voucher  to each participating household to thank them for their participation in all SAPRIN-related activities. Responses to questions were electronically captured in secure, on-premise databases with role-based security. Personally identifiable data were sequestered in separate database tables with restricted access, and all analytic data sets were pseudonymized prior to analysis. All study procedures were approved by Limpopo, Mpumalanga, and KwaZulu-Natal’s provincial Department of Health Research Ethics Committees (RECs), the University of KwaZulu-Natal’s Biomedical REC, the University of the Witwatersrand’s Human REC , and the University of Limpopo’s Turfloop REC.The adaptation of the existing SAPRIN surveillance program was discussed with and approved by each nodes’ existing community advisory groups prior to the finalization of the study protocol. The results of the studies were routinely shared with the community through a range of engagement activities that were conducted by the teams at each node.The data collected in the SAPRIN COVID-19 surveillance survey, including those reported in this paper, will be made available in pseudonymized form through the SAPRIN data repository .Between April 15 and December 24, 2020, AHRI  completed 14 waves of data collection, Agincourt  completed 12 waves of data collection, and DIMAMO (in Limpopo) completed 11 waves of data collection . These wDescriptive statistics for all respondent households with valid telephone numbers and members who consented to and completed an interview are shown in Respondents’ reported inability to access health care remained low and relatively stable over time. However, households reported that members missed daily medications in the past week at 52.9%  of interviews. Although rates of missed daily medications were stable over time at Agincourt and DIMAMO, they almost doubled at AHRI from 37.4% (315/843) in June to 64.9% (634/979) in July, as the epidemic arrived in the local area. The proportion of households that reported that members lost earnings due to COVID-19 regulations dropped substantially as the lockdown was reduced from Level 5 to Level 4. This proportion dropped again when the lockdown was lowered to Level 3 and remained steadily low thereafter. Finally, the proportion of individuals who screened positive for possible anxiety and depression fell over time at AHRI and DIMAMO and stayed low at Agincourt throughout the period studied.After accounting for study node and month of interview, and despite variation in household composition, no household characteristics were substantively associated with (1) respondents self-reporting satisfaction with their COVID-19–related knowledge; (2) residents leaving their homes in the week before the interview; or 3) residents avoiding travel or face mask use  and 3. H residentP=.55) but were less likely to have been unable to access medicine (P=.18) greater in households that received pension grants compared to those that did not receive pension grants  as the second epidemic wave spread across the country.These behavioral responses reflected local epidemic dynamics. The first national epidemic started in the Western Cape and spread first to the adjacent Eastern Cape before spreading to the densely populated Gauteng province and urban eThekwini in KwaZulu-Natal and then finally reaching the rural eastern and northern areas of South Africa, which were analyzed in this study. The second wave began in the Eastern Cape during November before spreading nationwide. The patterns of rapid behavior change in the face of a rising epidemic wave seen in this study were congruent with those seen worldwide. However, there are limited data on behavior in low- and middle-income countries either after first waves have receded or during subsequent waves. The impact of regulations and epidemic trajectories on travel is particularly pertinent in our study settings, as medium- and long-distance circular labor migration to urban areas is highly prevalent and vital to the economic well-being of rural and periurban South African households . Bans onP=.008) and nonsignificantly more likely to report anxiety . Comparisons are complex, but our findings align with national South African data, which suggest that COVID-19–related mental health impacts were more limited in low-income and rural areas [Third, our mental health findings are reassuring. At all three nodes, we observed declines over time in the prevalence of depression and anxiety symptoms . The prevalence of such symptoms was notably higher at the DIMAMO periurban node in Limpopo earlier in the year. This higher prevalence perhaps reflected concerns of being at greater risk of SARS-CoV-2 infection due to respondents’ proximity to the nearby city of Polokwane. However, it was encouraging that even when the COVID-19 epidemic arrived at AHRI in KwaZulu-Natal during July 2020, depression and anxiety prevalence did not increase, though the uptick in these rates that occurred late in the year as the second wave arrived at Agincourt in Mpumalanga may be concerning. Mental health concerns were much more prevalent at some sites than at others, particularly earlier in the year, but it is also notable that households with postsecondary-educated members were significantly more likely to report depression  and that a member had wanted but was unable to access health care in the 7 days before the interview 6.7%  of the time. These levels are similar to those of other South African surveys . NotablyFinally, we found that households with higher numbers of older members and pension recipients reported more unmet health care needs but fewer instances of missed daily medication or lost earnings. South African noncontributory pensions—broad national government support schemes that are often the largest household income source in these relatively rural settings with very high unemployment rates—have previously been linked to positive physical and mental health outcomes ,48. Our This study presents an overview of key insights across time from multiple sites across South Africa. However, there are several additional analyses that could further contextualize our findings. First, data can be longitudinally analyzed at the household or individual level to evaluate trajectories of behavior and impact as the COVID-19 epidemic continues to affect rural and periurban environments. For example, it will be important to evaluate the impact of new government policies, such as the ending of temporary increases in noncontributory grants. Second, these behavior-related data can be linked to COVID-19 symptoms and individual and population health outcomes to evaluate how risk perceptions and reactions are associated with health outcomes. Third, a more in-depth analysis of how household members’ historical and current age, gender, employment status, and migration composition, as well as preexisting comorbidities, affect the impacts of COVID-19 and NPIs will help identify those who are most in need of support during such crises. The ongoing SAPRIN COVID-19 surveillance program will enable the longitudinal measurement of these factors throughout the epidemic’s course.This study has limitations. As with all observational studies, the generalizability of our results to those outside our study population—in this case, households in rural and periurban areas of eastern South Africa—is uncertain. This concern was tempered by our ability to compare and combine data across multiple sites and compare our results to those of other studies on the COVID-19 epidemic’s impact in South Africa and Sub-Saharan African. Additionally, while household cellphone ownership was high, there was evidence that lower-wealth households in these areas were somewhat less likely to participate in the survey. Although nonrandom response may have affected prevalence measures, it should have very limited scope to affect the trend measures we focused on. Further, our data were self-reported and thus represented perceived needs and impacts, and changes in reported behavior may have reflected desirability biases. However, even with such biases, our findings provide insight into the perceptions and lived experiences of these communities. Comparing our findings to digital data sources can help alleviate such biases. Finally, we did not have data on identical questions from the pre-epidemic period; however, we were able to include similar information on many topics from earlier surveillance studies.This study also has several strengths, including a clearly defined sampling base, high response rates, low attrition rates, frequent follow-up, and linkages to pre-epidemic household data. Although we were not able to interview the same people in every survey wave, our longitudinal household cohort design, which allowed for repeated interviews with the same households over multiple months, reduced the risk of confounding by time-invariant household factors that could have arisen if we had used multiple cross-sectional surveys. Many of these benefits arise from the nature of the existing SAPRIN surveillance infrastructure, which reinforces the importance of long-term, population-based surveillance systems that collect social, demographic, and health data. This study demonstrates that surveillance systems can be rapidly repurposed to respond to emergency health needs, including (1) rapid pathogen data acquisition; (2) the identification of susceptible populations; (3) the assessment of behavioral and biomedical interventions; and (4) the development of mitigation strategies .SAPRIN nodes have been working with their local communities for 20-28 years. Such long-term engagement promotes deep understanding and community buy-in, which in turn enables rapid implementation and sustained, high-intensity follow-ups with minimal dropout. The network nature of SAPRIN also allowed each node to flexibly implement an overarching protocol. Furthermore, the use of telephonic call centers at each node allowed for rapid survey rollout that was based on previously provided informed consent for personal calls and substantially reduced the risk of SARS-CoV-2 infection among study staff and research participants. Since these call centers employed locally recruited staff, we were able to reach population segments that web-based surveys  and even random-digit dialing approaches  struggle to capture. Additionally, we could link self-response survey data to other data sources within the SAPRIN databases. These include the previously collected sociodemographic data used in this study and biological samples that were collected as part of the COVID-19 surveillance project. SAPRIN data can also be linked to data on public sector health care use and laboratory test results through memoranda of understanding with government departments. SAPRIN’s ongoing expansion will also allow comparisons with well-characterized urban sites to be made in the future.South Africans in three rural and periurban areas were largely willing and able to comply with national government regulations and recommendations regarding social interaction and other risk behaviors related to COVID-19, despite limited resources and the substantial economic need to travel. This rapid uptake of preventative behaviors reflects the clarity of government messages and the population’s willingness to comply with such measures, even in settings where enforcement measures were limited. Even as official NPIs were relaxed, the arrival of the epidemic in local areas led to further self-imposed behavioral restrictions, several of which led to difficulties in accessing health care. However, the economic and mental health effects of NPIs continued to decline as the measures were eased. Our findings highlight the importance of monitoring the possibly deleterious secondary impacts of NPIs in epidemic situations. Our results reinforce the principle that NPIs should be adjusted based on epidemic cycles and show that mitigation measures will be required to combat anticipated and unanticipated secondary impacts. All of these factors should be considered when setting, adjusting, and relaxing NPIs in low-income settings, especially as urgently established national policies give way to differentiated, decentralized approaches across diverse subnational environments.


In wire arc additive manufacturing of Ti-alloy parts (Ti-WAAM) gas metal arc welding (GMAW) can be applied for complex parts printing. However, due to the specific properties of Ti, GMAW of Ti-alloys is complicated. In this work, three different types of metal transfer modes during Ti-WAAM were investigated: Cold Metal Transfer, controlled short circuiting metal transfer, and self-regulated metal transfer at a direct current with a negative electrode. Metal transfer modes were studied using captured waveform and high-speed video analysis. Using these modes, three walls were manufactured; the geometry preservation stability was estimated and compared using effective wall width calculation, the microstructure was analyzed using optical microscopy. Transfer process data showed that arc wandering depends not only on cathode spot instabilities, but also on anode processing properties. Microstructure analysis showed that each produced wall consists of phases and structures inherent for Ti-WAAM. α-basketweave in the center of and α-colony on the grain boundary of epitaxially grown β-grains were found with heat affected zone bands along the height of the walls, so that the microstructure did not depend on metal transfer dramatically. However, the geometry preservation stability was higher in the wall, produced with controlled short circuiting metal transfer. In recent years, WAAM of Ti-alloy parts (Ti-WAAM) has been rapidly developed according to the rising demand of cheap high-scale Ti-alloy parts. One of the most required Ti-alloys is Ti-6Al-4V, which is broadly used in the aerospace industry due to its low density, high strength-to-weight ratio, outstanding corrosion resistance, and good performance at an elevated temperature ,2,3. It Generally, Ti-WAAM is performed using either gas tungsten arc welding (GTAW) ,6,7,8 orUsually GTAW- and PAW-WAAM parts have no defects, so researchers are concerned with resolving the issues regarding geometric parameters or the microstructure using different post-processing or interpass technologies, namely hot isostatic pressing , rollingCompared to GTAW and PAW, gas metal arc welding (GMAW) has a decisive bene-fit—in GMAW the arc exists between the substrate and the filler wire, which is fed coaxially to the arc. This advantage drastically increases the overall flexibility of WAAM with GMAW, which is why this method is regularly used in WAAM of steels  and Al-aHowever, GMAW of Ti-alloys at relatively low currents (lower than 300 A ) has beeVarious attempts have been made to stabilize the metal transfer during GMAW of Ti-alloys. Zhang et al. developed a modified active control of pulsed GMAW for Ti-alloy wire metal transfer and significantly increased the metal transfer process stability compared to the conventional-pulsed GMAW . Otani dThus, GMAW is utilized in Ti-WAAM less frequently than GTAW or PAW due to complications regarding the metal transfer of the Ti-wire during manufacturing. Several studies did not report any issues concerning metal transfer during Ti-WAAM using CMT ,36. NeveThe aim of the present work was to investigate and compare three metal transfer modes in Ti-WAAM: CMT mode, controlled short-circuiting (CSC) mode, and a conventional mode at a direct current with a negative electrode (DCEN). The investigation was provided with the acquired current and voltage waveforms and transfer process high-speed video analysis. Moreover, the influence of the transfer mode on the geometrical parameters and the metal properties of the manufactured structures was evaluated. The significance of the present research is highlighted by the opportunity provided to obtain clearer information on the arc wandering phenomenon during GMAW-based Ti-WAAM.The walls were printed using the WAAM system which was composed of a Motoman MH24 robot  and an Alpha Q 552 welding power source  or a TransPuls Synergetic 3200 welding power source . Ti-6Al-4V alloy was used as both the filler wire and the initial substrate. The filler wire had a diameter of 1.2 mm and the substrate was 8 mm thick. Pure argon (99.99%) was used as a shielding gas, the metal was deposited inside a non-vacuum sealed box with a controlled argon atmosphere.Three walls were printed using CMT, CSC, and DCEN metal transfer modes. CMT was performed using Fronius TransPuls Synergetic 3200 power source, CSC and DCEN were performed using EWM Alpha Q 552 power source. The travel speed was 20 cm/min, the wire feed rate was 4.5 m/min and these parameters did not vary between the printed walls in order to compare the metal transfer modes. The average current and average voltage values are shown in The metal transfer process was shot using a Chronos high-speed video camera  and a backlight 40 W LED matrix. The current and voltage waveforms were captured at a frequency of 100 kHz using a GW Instek GDS-71104B oscilloscope and a 300 μOhm shunt when the Fronius TransPuls Synergetic 3200 power source was used. In the case when the EWM Alpha Q 552 power source was used, voltage and current were recorded from the power source output information port at a frequency of 24 kHz. Captured waveforms were processed via Matlab and noises filtered using a median filter with a 15 samples window and a moving average filter with a 5 samples window.Transverse sections of walls were prepared using the Buehler grinding and polishing system. Grinding was carried out in three steps with a gradual increase in the grit of the SiC sandpaper: p240 was used for 2 min, p800 for 2 min, and p1000 for 3 min. The sand-paper and the sample holder were rotated in controversial directions, the rotation speeds of the sandpaper and the sample holder were 220 rpm and 160 rpm respectively. The load for pressing the samples was 30 N.Polishing was carried out in four steps with a gradual decrease in the diamond sus-pension grain size: 9 μm and 6 μm suspensions were used with an NX-MET X500 pol-ishing cloth, 3 μm and 1 μm suspensions were used with the NX-MET X220 polishing cloth. Polishing time was 5 min for each suspension, the polishing cloth and the sample holder were rotated in the same direction. The rotation speeds of the polishing cloth and the sample holder were 120 rpm and 160 rpm respectively, the load for pressing the sam-ples was 25 N.The cross sections were etched using a solution of 96 mL of distilled water, 2 mL of hydrofluoric acid, and 2 mL of nitric acid; the samples were kept in the etchant for 75 s. A Leica DMI 5000M metallographic microscope was used to obtain the microstructure images.Three CMT transfer cycle waveforms are represented in The second frame in Despite the fact that the processes occurring at the anode are considered much less often than the processes occurring at the cathode , the disRare spattering can be observed during the CMT process , spatterThree CSC transfer cycle waveforms are represented in Three DCEN transfer cycle waveforms are represented in However, as shown in Full high-speed videos of the CMT, CSC, and DCEN metal transfer processes are available as the The effective wall width considerably differs between walls . It is pHere, the wall width efficiency is influenced by the wall waviness, which depends on the heat input distribution. Heat input distribution varies during GMAW due to arc wandering, and its effect was greater in the CMT process as a result of the extended arc length. The DCEN wall inconsistency is a consequence of the higher heat input into the anode , which iEach wall microstructure in The microstructure images a,c,e repGrain boundary α forms at the prior β-grain boundary and there is a preferred orientation between the grain boundary α and the primary β-grain ,42, whicBlack and white bands that are observed on each wall microstructure  are heatIn summary, it can be noted that there is no critical difference in the printed walls microstructure, thus the metal transfer at the investigated intervals of the transfer parameters primarily influences the geometrical properties of the manufactured structures. Therefore, although the other works on Ti-WAAM are generally based on the CMT transfer process ,36,37, tDuring CMT, the burning arc at the ignition start misses the wire tip, which occurs almost in every cycle and can significantly affect the heat input distribution. Such a phenomenon can be explained by the cumulative effect of the low wire tip temperature and the refractory properties of Ti, which results in insufficient anode vaporization; the lack of anode vaporization develops in the diffusive arc burning mode until the wire tip is heated enough to produce the anode spot. The discovered effect tends to play a crucial role in arc burning stability during CMT.The effective wall width considerably differs between walls, printed using different transfer modes. The width efficiencies are 0.62, 0.76, and 0.56 for the CMT, CSC, and DCEN walls, respectively. The wall width efficiency is influenced by the wall waviness, which depends on the heat input distribution. Heat input distribution varies during GMAW due to arc wandering, and its effect is greater in the CMT process as a result of the extended arc length. The DCEN wall inconsistency is a consequence of the higher heat input into the anode, which in the case of DCEN is the wall. The relatively high CSC wall efficiency is the result of the low arc length and sufficient heat input, produced during the arc burning phase of the metal transfer cycle.There was no critical difference found in the printed walls microstructure, thus the metal transfer at the investigated intervals of the transfer parameters primarily influences the geometrical properties of the manufactured structures.Owing to its width efficiency and sufficient arc stability, the CSC transfer process was found to be the most suitable for thin-walled structure printing among the three studied metal transfer modes. The next study will be dedicated to further development of CSC metal transfer at higher process rates.In this study, three metal transfer modes were applied in GMAW-based low current Ti-WAAM, namely CMT, CSC, and DCEN. The transfer modes were investigated, results of their application were compared, and the conclusions are as follows:


Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected.Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Literature search revealed developments and usage of expression systems in AAB.• Only recently 2 regulatable plasmid systems became available for only 2 AAB genera.• Further regulatable expression systems for AAB are in sight.•  Acetic acid bacteria (AAB) are a group of obligately aerobic Gram-negative bacteria that exhibit a unique form of metabolism by which they typically partially oxidize a variety of substrates such as sugars or ethanol by membrane-bound dehydrogenases (mDHs) and produce acetic acid. AAB are already used for a long time and they are very important for the production of foods and beverages such as vinegar, kombucha, kefir, and other products. Several reviews on AAB are already available that provide overviews, discussions and outlooks on issues related to their taxonomy, physiology and biochemistry, fermentation (foods and beverages), acetic acid resistance, production of exopolysaccharides including bacterial cellulose, pyrroloquinoline quinone (PQQ)-dependent dehydrogenases, oxidation of carbon sources and alcohols, nitrogen fixation, metabolic engineering, biotechnological and industrial applications, as well as their use as microbial biosensors . In this first literature filtering step, the single AAB genus word-based searches in the WoSCC database revealed that the top 3 AAB genera with by far the most publications were Acetobacter , Gluconobacter  and Glucon(o)acetobacter , respectively. These top 3 genera were followed by Acidiphilium (345), Komagataeibacter (229), Roseomonas (177), Asaia (175), Acidocella (62) and Acidomonas (33). As outlined below, some genera names were introduced only recently and many of the included species had a different genus name before. Together, these 9 AAB genera already represent 97.6%  of all  non-redundant publications somehow related to one or more out of the 49 AAB genera. We then selected the 6097 publications from the year 2020 backward to 1973, the year of the first published successful DNA cloning experiments in Escherichia coli  database. Currently, for the Acidomonas was proposed to include a group of acidophilic, facultatively methylotrophic bacteria . As a consequence of these changes and updates, Acetobacter diazotrophicus, A. europaeus, A. hansenii, A. liquefaciens, and A. xylinum  were included in the genus Gluconacetobacter with the respective changes in nomenclature: Gluconacetobacter diazotrophicus, Ga. europaeus, Ga. hansenii, Ga. liquefaciens, and Ga. xylinus, respectively. Furthermore, in the following years, a phylogenetic duality in the new genus Gluconacetobacter was found. The Ga. liquefaciens group and the Ga. xylinus group could be phylogenetically, phenotypically and ecologically distinguished from each other at the generic level. This resulted in the creation of the new AAB genus Komagataeibacter based on 16S rRNA gene sequences and the transfer of several new combinations including Ga. xylinus  to Komagataeibacter xylinus on the basis of taxonomic characteristics . Initially, in 11 studies, several plasmids were constructed and tested before a major expression plasmid family was established in Gluconobacter in 2006.Most studies reporting on the construction and usage of expression plasmids in AAB were found for the genus E. coli / Gluconobacter shuttle vector was published in 1985 by Teruhiko Beppu and co-workers, who in parallel were also working on shuttle vectors for Acetobacter (see Acetobacter section). This first shuttle vector for Gluconobacter was constructed by ligation of cryptic plasmid pMV201 (2.5 kb) found in G. suboxydans IFO 3130 with the E. coli plasmid pACYC177 (3.9 kb) carrying the p15A ori. The resulting chimeric plasmid pMG101 (6.2 kb) carries an ampicillin (Amp) resistance gene and replicates in G. suboxydans as well as in E. coli  and Q (pKT230/231) was studied. A gentamycin (Gm) resistance-encoding pRK290 derivative  was constructed and suggested to offer considerable potential as a versatile gene delivery system for Gluconobacter -dependent genes homologous to the E. coli lacI, lacZ, and lacY genes. The Tn951-based β-galactosidase activity was found to be induced 4-fold to 6-fold by IPTG in Gluconobacter, which suggested the synthesis of a functional LacI repressor in Gluconobacter, yet the repression was leaky in the absence of IPTG and the induced LacZ activity less than 5% compared to fully induced levels in E. coli. Since in E. coli the lac operon displays reversible cAMP-dependent catabolite repression with glucose, cAMP was exogenously added (100 μM) to the Gluconobacter culture. cAMP did not result in any detectable increase in LacZ activity, suggesting that a catabolite repression-like phenomenon was unlikely to be the cause of the poor LacZ activity in Gluconobacter if the assumption holds true that cAMP can be transported into the cell at levels sufficient for physiological effects to be observed. With the genome sequences available today we know that a CRP gene appears to be absent in Gluconobacter. The low levels of LacZ activity could be explained by inefficient recognition of the heterologous Tn951 promoter in Gluconobacter  can be transferred to G. oxydans IFO 3293 and plasmid pSUP301 (5 kb) from E. coli  by conjugal transfer, maintained very stably without antibiotic selection, and did not inhibit the growth or 2-keto-l-gulonic acid productivity of producer strains derived from G. oxydans IFO 3293. pGE1 could also be shortened to a 9.8-kb plasmid termed pGE2. pGE1 could be introduced in 6 Gluconobacter and 4 Acetobacter strains out of 6 and 28 strains tested. Thus, it showed a broader host-range than the shuttle vector pMG101 introduced above, which showed a limited host-range even in Gluconobacter. pGE1 was considerably stable in the transconjugants after two cycles of cultivation without kanamycin, except in G. frateurii IFO 3271. The usefulness of pGE1 as an expression vector was confirmed by subcloning the membrane-bound l-sorbosone dehydrogenase (SNDH) gene of A. liquefaciens IFO 12258 and its expression in G. oxydans IFO 3293 derivatives. Using resting cells, pGE1 derivatives with the SNDH gene, however, did not lead to a higher 2-keto-l-gulonic acid production than the pVK102 derivative with the SNDH gene  was constructed from the endogenous cryptic plasmid pGO3293S (9.9 kb) found in G. oxydans T-100 was ligated with plasmid pHSG298 (2.7 kb) carrying pBR322 ori and the resulting chimeric plasmid pFG15A (7.3 kb) was confirmed to be also stable in G. oxydans G624 across several passages in a broth without kanamycin and had no effects on the l-sorbose production by the host  extracted from Rhizobium, Agrobacterium, and Pseudomonas species, respectively, was used in Gluconobacter to complement a Tn5-based pqqE-deficient mutant by expressing a genomic library fragment containing the pqqE gene of the PQQ coenzyme biosynthesis gene cluster pqqBCDE of G. oxydans ATCC 9937 .In another study, the broad-host-range vector pSUP104, which includes the mobilization and replication functions of the IncQ plasmid RSF1010 and was originally tested in Gluconobacter is the shuttle vector pSA19 already developed and introduced for use in Komagataeibacter in 1994 (see Komagataeibacter section). The feasibility of using pSA19 was also demonstrated for Gluconobacter by expressing the xylitol dehydrogenase (XDH) gene of Morganella morganii from the promoter Plac  genes sldSLC in G. frateurii THD32 to analyze SboA enzyme activity and the role of the putative transcriptional regulator SboR in the regulation of sldSLC expression  the SboR-repressed promoter of the sboRA operon seems not suitable to be used for a regulatable expression system in Gluconobacter.Another expression plasmid used in Gluconobacter, another shuttle plasmid was created and tested. The cryptic endogenous plasmid pAG5 (5.6 kb) found in G. oxydans IFO 3171 was ligated with pUC18 and the chimeric vector was termed pSG8  in the absence of ampicillin. Plasmid pSG6 was used for expression of the endogenous cyanide-insensitive quinol oxidase genes cioAB in the wild type G. suboxydans IFO 12528  isolated from Gluconobacter studies reporting recombinant gene expression; thus, it is the major expression plasmid lineage used in Gluconobacter and it also exhibits the most use cases in AAB in general  found in two G. oxydans strains and pUC18 or pUC19 carrying pBR322 ori. The chimeric plasmids are selectable by conferring ampicillin resistance, are compatible with the pBBR1MCS family and double selectable except with pBBR1MCS-4 also carrying ampicillin resistance. In the first case, the par and rep loci of pGOX3 from G. oxydans DSM 2003 were amplified as a 2.3-kb fragment and cloned into pUC18, resulting in the 5 kb shuttle vector pZL1  genes and two l-sorbosone dehydrogenase (SNDH) genes from Ketogulonicigenium vulgare in G. oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to l-sorbose. As the production of the vitamin C precursor 2-keto-l-gulonic acid from d-sorbitol involves three sequential oxidation reactions, the spatial proximity of the corresponding dehydrogenases appeared to be useful to enhance the production. Therefore, with a series of linker peptides, SDH-SNDH fusion enzymes were tested and for a selected SDH-SNDH fusion the pqqA gene alone or the pqqABCDE gene cluster for PQQ biosynthesis was additionally included on the pGUC plasmid. Additional expression of the PQQ biosynthesis gene(s) enhanced cell growth and with the stepwise metabolic engineering of G. oxydans, the final 2-keto-l-gulonic acid titer was improved 8-fold (39 g/L) compared to that obtained with the independent expression of the dehydrogenase genes  and moderate (PGOX0452), while the intrinsic promoter Plac of pBBR1MCS-2 appeared rather weak in G. oxydans 621H. In another approach to identify strong promoters, chromosomal DNA of G. oxydans DSM 2003 was randomly digested and DNA fragments were inserted into pBBR1MCS-5-gfp carrying the promoterless green fluorescent protein gene as a reporter  than PtufB from G. oxydans (1.39 U/mg) when assayed with pBBR1MCS-5 and the ndh gene encoding the type II NADH dehydrogenase as a reporter.With the aim to provide effective PB932_2000 was identified in G. oxydans WSH-003. As assessed by analysis of pBBR1MCS-2-based egfp expression and qRT-PCR analysis of egfp mRNA, the strength of PB932_2000 in G. oxydans WSH-003 was at least 2-fold higher than that of PtufB from G. oxydans and at least 4-fold higher than that of PtufB from E. coli. In a further study the aforementioned G. oxydans promoters PtufB, PGOX0169, PGOX0264, and PGOX0452 were assayed for their relative strength in G. oxydans 621H using pBBR1MCS-5 and gfp as reporter gene  and the inositol dehydrogenase gene (Pidh) were analyzed using pBBR1MCS-5-based pJV17 derivatives carrying the E. coli lacZ reporter gene. According to the β-galactosidase assays, both promoters were practically not active in E. coli, while they showed good activity in G. oxydans grown on d-mannitol, d-sorbitol or d-glucose as carbon source. Padh led to similar LacZ activities with all three substrates and was generally more active than Pidh, which led to weak LacZ activity on d-mannitol and appeared to be repressed 100-fold in the presence of d-glucose when compared to d-sorbitol.In contrast to the previous studies searching mainly for strongest promoters, another study aimed to develop a new vector for successful expression of genes encoding membrane-bound dehydrogenases in Gluconobacter. In an early study analyzing the maltose-oxidizing ability of a G. oxydans strain, the effect of a single amino acid substitution on the substrate specificity of the PQQ-dependent glucose dehydrogenase (mGDH) was tested by chromosomal insertion due to the lack of a suitable expression plasmid for Gluconobacter at that time  cycle of G. oxydans 621H needs to be completed by a succinate dehydrogenase and a succinyl-CoA synthetase, since these two TCA cycle enzymes are absent. These requirements were addressed by combining the chromosomal insertion of several heterologous genes and deletion of the two glucose dehydrogenase genes to eliminate the periplasmic and cytosolic oxidation of glucose to gluconate  selected on the basis of RNAseq data for their suitability to integrate and express the fructose dehydrogenase (FDH) genes fdhSCL from G. japonicus under the control of the strong promoter PGOX0264 for the oxidative biotransformation of fructose to 5-ketofructose (5-KF)  also plays an important role. According to the G. oxydans 621H genome sequences, a CRP appears to be absent   after the stop codon of the reporter gene, strongly reduced the maximum of the resulting reporter activity. In future studies, systematic analysis of such and other sequence alterations including terminators and orientations of neighboring genes  may reveal specific rules that should be considered for successful construction and establishment of tight and regulatable heterologous expression systems in Gluconobacter or in AAB.Until now, an BAD Fig. a. In the Schleif . Additiol-arabinose concentrations required for highest induction in G. oxydans were much higher than the ones typically used for E. coli. Attempts to increase the arabinose sensitivity of the araC-ParaBAD system by expression of the l-arabinose transporter gene araE in tandem with araC from the promoter of araC had a strong negative effect on the growth of G. oxydans and the expression performance . Thus, before using AraE to hopefully increase the sensitivity of the araC-ParaBAD system toward l-arabinose, the strong negative effect of araE expression should be understood and overcome. Therefore, conditional and tuned expression of araE using the araC-ParaBAD system itself could help to clarify the negative effects of araE expression in G. oxydans. Likewise, araC-ParaBAD-dependent conditional expression could also help to express genes for other “difficult” transporters and (membrane-bound) enzymes in AAB.The Komagataeibacter reporting on expression plasmids, among which 5 major lineages can be identified. Almost all studies employed electroporation as the method of choice for transfer of plasmids into Komagataeibacter, whereas conjugation was rarely used  and analyzing the number of transconjugants  and cellulose-negative (Cel-) mutants of K. xylinus the broad-host-range cloning vector pVK100 and cosmid vector pRK311 were used to prepare gene libraries of K. xylinus to search for genes expressed from their native promoters and complementing the Cel- phenotype  with several antibiotic resistances into bacterial cellulose-producing K. xylinus, was used for the construction of a genomic library  was found. The latter was used to construct two shuttle plasmids by ligating the small cryptic plasmid with the E. coli plasmids pUC18 and pUC19 at the SstI sites to obtain pUC18-824 and pUC19-824. These shuttle plasmids confer ampicillin resistance and also serve as expression vectors due to the presence of Plac from E. coli which is actively transcribed in K. xylinus. Cloning of the single bcs genes expressed from Plac allowed specific complementation of different Cel- mutants. Additionally, replacing the native bcs promoter Pbcs on the chromosome with the heterologous promoters Plac or Ptac reduced the cellulose synthase activities of cells to -60% and -85% of the level of the parental strain, demonstrating that the promoter Pbcs is quite strong and useful for high expression of desired target genes. In a later study on the cellulose-synthesizing operon acs in another K. xylinus strain, the cosmid vector pRK311 already mentioned above was used to express the single gene acsD from Ptac for recovery of normal cellulose production in an acsD mutant  fused with pUC18 or with pTrc99A, the plasmids pUF106 and pTA99 were constructed to obtain shuttle vectors Bacillus and a mutated variant (H131F), which is inactive but still binds to cellulose, to study their effects on cellulose production by K. xylinus sucZE3 from Zymomonas mobilis together with the secretion-activating factor gene zliS and the B. subtilis levansucrase gene sacB containing a mutation causing decreased levan-forming activity were cloned into pSA19 under the control of Plac to study cellulose production by Komagataeibacter from sucrose, which is the most suitable carbon source for the economical production of bacterial cellulose Leuconostoc mesenteroides was expressed with pSA19 using several promoters  to improve cellulose production in K. xylinus  gene from (iv)bcs from a K. xylinus strain instead of Plac to study this bcs promoter region  of the cellulose synthase operon (v)K. xylinus under control of Plac K. xylinus and functional analysis of the ORF2 polypeptide involved in cellulose synthesis, the ORF2 sequence plus a kanamycin resistance cassette was cloned into pSA19 K. xylinus mutants with disrupted aceR and aceQ, these genes involved in acetan biosynthesis were cloned into pSA19 for expression K. xylinus in which the carboxymethylcellulase gene cmcax was disrupted by an ampicillin resistance cassette, the cmcax gene was expressed in trans using the pSA19 backbone carrying a kanamycin resistance cassette as an additional selection marker  from a cellulose-producing Komagataeibacter is the broad-host-range plasmid pBBR122 introduced above in the Gluconobacter section. With this plasmid, the gene of the bacterial hemoglobin from Vitreoscilla (VHb) was expressed driven by the constitutive bla promoter in cellulose-producing K. xylinus , the gene of which was constitutively expressed from Plac using pBBR122  synthesis was expressed from the constitutive promoter Pbla in K. xylinus with the aim to introduce GlcNAc residues by cellulose synthase and produce a chimeric polymer . pMV24 was used to study the regulation and function of the genes ginI, ginA and ginR of a quorum-sensing system from a K. intermedius strain producing three different N-acylhomoserine lactones  from their native promoters  with the sequence for an octapeptide FLAG epitope tag allowing detection via immunoblotting  toolkit, a resource for constructing optimal plasmid vectors based on a minimized backbone and three interchangeable modules to design a compilation from several origins of replication, diverse antibiotic resistance markers, and a cargo of interest, flanked by uncommon restriction sites  or N-acyl homoserine lactone (AHL) were tested. From these, the AHL-inducible constructs showed higher induction and lower leakiness than the ATc-induced constructs, yet the induction fold-changes were only up to 5-fold under the conditions tested  and mRNAs to facilitate mRNA translational regulation and a sRNA targeting UDP-glucose pyrophosphorylase (UGPase) mRNA were co-expressed from a SEVA plasmid with pBBR1 ori in response to the AHL inducer to inhibit UGPase mRNA translation. UGPase catalyzes the production of UDP-glucose, the substrate for cellulose synthesis, and is encoded by a single gene in the genome, allowing knockdown by a single sRNA. This system was found to be highly efficient, as cellulose production was suppressed completely upon full induction and could be fine-tuned using different concentrations of AHL. With E. coli Hfq and broad-host-range backbone, the system was engineered to be a general platform for targeted knockdowns in Komagataeibacter and other bacterial species independent from the host Hfq by adding new sRNA sequences to the plasmid making the construct easily modifiable for other mRNA targets  homoserine lactone in the transformed K. rhaeticus strain (sender). In another K. rhaeticus strain carrying the luxR-Plux system (receiver), the signal molecule was sensed, affecting the expression of the mRFP gene encoding the fluorescence reporter protein. It was demonstrated that communication can occur both within and between growing pellicles in mixed cultures of the two strains.The SEVA-based lux, Ptet, ParaBAD), natural and synthetic terminators and ribosome binding sites as well as protein degradation tags in K. xylinus, K. hansenii, and K. rhaeticus iGEM  with a catalytically inactive Streptococcus pyogenes Cas9 protein (dCas9) was also tested as a tool to readily knock down the expression of a target gene by blocking transcription. Therefore, the dCas9 gene fused to a 3xFLAG tag DNA sequence was expressed by the strong promoter J23104 together with a single guide RNA (sgRNA) gene under the control of the native tracrRNA promoter from S. pyogenes. Alternatively, the sgRNA gene was positioned without promoter just downstream of the dCas9-3xFLAG gene, thus forming an operon. The mentioned constructs were cloned into a single pSEVA331Bb plasmid backbone. When targeting the 5’-ends of the acsAB gene and of the acsD gene of the endogenous acs operon for cellulose synthesis in K. hansenii by two different sgRNAs, a more than 2-fold decrease in acsAB expression and 15% reduced cellulose yield were observed, while for acsD no significant change in expression was observed.SEVA-based plasmids were also used to characterize 11 constitutive promoters, 3 inducible promoters  in a GFP reporter library  than that of wild-type K. xylinus. In static conditions 5.3 g/L cellulose was reached, demonstrating that reinforced metabolic flux toward bacterial cellulose through modified gene expression represents a practical approach for the improvement of bacterial cellulose production. By another strategy, which included the deletion of a PQQ-dependent glucose dehydrogenase gene and co-expression of the glucose facilitator gene glf from Zymomonas mobilis and the endogenous glucokinase gene glk from K. xylinus, a somewhat higher cellulose titer (5.9 g/L) was obtained with K. xylinus in a specific growth condition  sites, all flanked by the targeted gene-flanking regions. A second expression plasmid, pFLPGX, containing lacIq, a pBBR1 ori flanked by FRT sites, a spectinomycin resistance gene, and the FLP recombinase gene flp controlled by LacIq-dependent Ptrc, was also constructed via Gibson assembly. pFLPGX with the same ori as pRedGX but a different resistance gene was used to eliminate the λ-red plasmid by antibiotics selection and to produce FLP recombinase, which catalyzes the reciprocal recombination between the FRT sites introduced into the chromosome for in-frame gene deletion and also between the FRT sites in the flp plasmid (FRT-pBBR1 ori-FRT) for self-elimination of the flp plasmid. Using the λ-red and the flp expression plasmids, four putative glucose dehydrogenase genes were deleted in K. xylinus, resulting in the identification of one of these genes as being responsible for gluconate formation from glucose. In the respective mutant, the glf and glk genes were constitutively co-expressed from a plasmid with a pBBR1 ori and a kanamycin resistance gene. The results obtained indicated that glucose was transported into the cell by the facilitator protein and glucokinase further increased the production of cellulose , glucose-6-phosphate isomerase and 6-phosphogluconate dehydrogenase encoded by pgi and gnd have been predicted as novel overexpression targets for the enhanced CNF production  have been constructed by ligation of cryptic plasmid pMV102 endogenously present in A. aceti subsp. xylinum NBI 1002 with the E. coli plasmids pACYC177, pBR322, or pBR325. The initial size of chimeric plasmids (>6 kb) was reduced by a series of consecutive digestion and ligation steps delivering pMV329 with only 3.4 kb as the smallest vector A. polyoxogenes fused to the short N-terminal β-galactosidase peptide was produced in an A. aceti mutant lacking the enzyme activity A. aceti obtained by chemical mutagenesis revealed three genes termed aarA, aarB, and aarC responsible for acetic acid resistance. Gene aarA was identified to encode a citrate synthase aarC gene expressed from the pMV24 backbone was found to functionally complement an aarC gene disruptant mutant of A. aceti, which is defective in acetate assimilation A. pasteurianus was cloned into pMV24 to corroborate a second transcriptional start by primer extension analysis  gene of (v)A. pasteurianus mutant was transformed with a pMV24-based genomic library and the recombinants were screened for ethanol resistance. The his1 gene encoding histidinol phosphate aminotransferase was shown to be responsible for the suppression of the ethanol sensitivity of the mutant. This effect was dependent on the higher copy number of his1 obtained with plasmid pMV24, as the single genomic his1 copy was not sufficient to enable ethanol resistance adhS gene encoding subunit III of the three-component membrane-bound alcohol dehydrogenase (ADH) from A. pasteurianus was expressed from pMV24 and restored the ADH activity of an adhS mutant deficient in subunit III gdhS gene for subunit III of the ADH from G. suboxydans, which strongly differs in its amino acid sequence from GdhS of A. pasteurianus, was expressed in the A. pasteurianus adhS mutant and could not restore the ADH activity est1 was shown to play a major role in ethyl acetate formation and its production was fully recovered in an A. pasteurianus est1 mutant by expressing an est1-containing DNA fragment from pMV24 A. aceti, pMV24-based overexpression of groESL encoding the chaperones GroEL and GroES, which are induced not only by temperature shifts but also by other stresses such as exposure to ethanol, conferred improved growth of A. aceti at increased temperature (42°C) and also supported the resistance against ethanol stress dnaKJ with its heat-shock promoter resulted in improved growth of A. aceti compared to the control strain at high temperature or in the presence of ethanol, suggesting a correlation to resistance against various stressors present during fermentation, although it did not increase the resistance to acetic acid A. aceti was increased when the aconitase gene was overexpressed from Plac using pMV24, showing that multiple copies of the aconitase gene, and accordingly an increase in aconitase activity, conferred acetic acid resistance A. pasteurianus, pMV24-based overexpression of the uvrA gene, whose product is involved in recognition and processing of DNA lesions, resulted in increased biomass formation in the presence of high acetic acid concentrations and resulted in upregulation of enzymes involved in ethanol oxidation and acetic acid tolerance. UvrA thus contributes to acetic acid tolerance by protecting genome integrity, thereby ensuring gene expression and metabolism  was used to construct the shuttle vector pMV24 by inserting pMV329 between the Acetobacter, the improved broad-host-range cloning vector pCM62 was used as an expression plasmid. It was originally developed for use in methylotrophs and other Gram-negative bacteria by combining the functions present in the minimal transferable and selectable RK2 replicon of the pTJS75 derivative pCM51 with the polylinker and pBR322 ori of pUC19 galE was expressed for complementation of a galE mutant of A. tropicalis, which was found to produce extracellular polysaccharide secreted into the medium instead of forming capsular polysaccharide involved in pellicle formation adhS of the PQQ-dependent alcohol dehydrogenase (ADH) from A. pasteurianus was expressed to complement an adhS disruptant mutant, which revealed that subunit III is essential for the formation of the active ADH complex besides subunit I and II A. tropicalis at higher temperatures were expressed to complement gene disruptant mutants obtained by transposon mutagenesis groESL were expressed in an A. pasteurianus groEL disruptant mutant for complementation and in the parental strain, which enhanced the resistance against 4% acetic acid and 5% ethanol aldFGH, which are indispensable for acetic acid formation from ethanol, and the isozyme genes aldSLC, whose mRNA levels were 10-fold lower during growth on ethanol compared to growth without ethanol, were expressed in A. pasteurianus fabG that may change the fatty acid composition and supports growth of A. pasteurianus in a low-nutrient environment were expressed. It enhanced growth of A. pasteurianus and the acetic acid production useful for cost-effective manufacturing of rice vinegar , pACT7 (3.9 kb) and its smaller BalI / PvuII-digested and self-ligated derivative pACT71 (3.3 kb)  genes adhAB from A. aceti in A. pasteurianus NP 2503 deficient in ADH  conferring kanamycin resistance, which can also replicate in E. coli , but no further work based on these plasmids was reported.In a study on the functions of the multiple heme Acetobacter study, the IPTG-inducible PT7-based Novagen vector pET-30a(+) with pBR322 origin was described to have been used in A. pasteurianus for inducible expression of the endogenous genes of DnaA- and DnaB-like proteins as well as the dnaA and dnaB genes from E. coli to study the replacement of the corresponding endogenous proteins expressed from the chromosome  or from another promoter included by the cloning procedure. Furthermore, plasmid backbone pBAD18 carrying pBR322 origin, the classical l-arabinose-dependent AraC regulator gene araC and the ParaBAD target promoter of AraC have been used in A. orleanensis for arabinose-inducible expression of asRNA fragments (121 nucleotides) which are antisense to the C-terminal coding sequence of the dnaA and dnaB gene sequences studied  from its native promoter Padh  and aldehyde dehydrogenase , PQQ biosynthesis genes (pqqAB or pqqABCDE), and combinations thereof, each under the control of the promoter Ptuf that was 1.8-fold stronger than PadhA in GFP reporter assays  thereby shortening the duration of the starting-up process from 116 to 99 h. This strategy is of significance for decreasing costs for producing high-strength acetic acid industrially and will also be useful for the production of other desired organic acids, especially those involving PQQ-dependent enzymes.In two recent Ga. diazotrophicus. Mostly, electroporation was used to transfer the recombinant DNA, yet conjugation was also used in some cases. The first recombinant DNA study in Gluconacetobacter was a complementation experiment in a levan synthesis-deficient mutant of the renamed A. diazotrophicus , recovered the abilities of the parental strain in the lsdA disruptant mutant.Taking into account the AAB genera and strain updates, as well as our exclusion criteria mentioned before, we found 10 studies using diverse plasmids for gene expression in Ga. diazotrophicus as an endophytic nitrogen-fixing plant-associated microbe is generally also interesting as a host to express genes encoding proteins that could enable control of pests attacking agricultural plants. In this context, the Bacillus thuringiensis cry genes are interesting since they encode δ-endotoxins exhibiting entomopathogenic activity. To test the control of coleopteran pests in sugarcane, the cry3A gene hooked in the transposon Tn5 was introduced into Ga. diazotrophicus by conjugation on the narrow-host-range suicide plasmid backbone pSUP1021 carrying the transfer origin oriT of plasmid RP4 and integrating into the genome  containing the mCherry gene under control of the promoter Ptac was used to analyze the colonization of Ga. diazotrophicus on two genotypes of elephant grass (Pennisetum purpureum), a perennial C4-plant employed for grazing, silage production or bioenergy, via seed coat and leaf spray  transport system comprising the three genes feuABC encoding a periplasmic-binding protein, a permease, and a traffic ATPase was analyzed earlier  and pPGH11 (R68.45 and pPGH1 from Pseudomonas) could be transferred into A. cryptum with frequencies of 1.8 x 10-2 to 9.8 x 10-4 transconjugants per recipient cell. In the transconjugants, the antibiotic resistances and the ability to degrade phenol were expressed. A. cryptum with pPGH11 grew with 2.5 mM phenol at a doubling time of 12 h and a yield of 0.52 g dry cell weight per g of phenol.A. multivorum was sequenced and analyzed to use its characteristics for the construction of a shuttle vector , but also with insects. The presence of AAB genera in insects , that typically use plant materials and sugar-rich matrices as food sources, is seen as roles of these AAB in exploitation of the food. Asaia was also found in the malaria mosquito vector Anopheles stephensi and is also present in and cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders  gene with its signal peptide sequence under the control of PnptII that was derived from a Wolbachia strain isolated from the nematode Dirofilaria immitis  of Pidh involved need to be unraveled as well as the performance and the suitability of Pidh for use in a regulatable endogenous expression system. The R. leguminosarum promoter of the nifHDK operon, which is activated under nitrogen-fixing conditions, was functional in plant-associated Ga. diazotrophicus in planta. Therefore, PnifHDK might also be used in nitrogen-fixing AAB ex planta to control gene expression in dependence of the available nitrogen sources  could possibly be transferred with all regulatory requirements to non-nitrogen-fixing AAB.Due to a deficiency in regulatable promoters, almost only constitutive promoters have been used in AAB. The greatest variety of endogenous, heterologous, and synthetic promoters functional in AAB and exhibiting weak, moderate, strong, or very strong activity is known for luxR-Plux system and the other one is a well-tunable l-arabinose-inducible araC-ParaBAD system with up to 480-fold induction  still leaky in Komagataeibacter for full functionality in any growth condition. In Komagataeibacter, an l-arabinose-inducible araC-ParaBAD system was also very leaky and thus induction fold-change was very low  (Fricke et al. G. oxydans 621H, the l-arabinose oxidation activity is high and contributes to the strong acidification of the growth medium by forming arabinonic acid causing a severe loss of reporter activities, while the d-arabinose oxidation activity is low (Fricke et al. d-arabinose-responsive AraC derivatives could circumvent the pH issue of l-arabinose induction and might probably also result in increased sensitivity toward arabinose concentrations. Therefore, engineered AraC mutant proteins with altered binding pockets which activate transcription in response to d-arabinose and not in response to l-arabinose could be tested (Tang et al. E. coli and were highly specific toward their target compound. That illustrates the potential of the AraC transcriptional regulatory protein for molecular sensing, reporting and target gene expression, which could possibly also be achieved and used in AAB.Until now, for AAB only two well-documented inducible heterologous expression systems are available for only two species from different genera. As described above, one is based on an AHL-inducible Komagataeibacter, is expected to speed up expression system development and testing or screening of such systems in even more AAB genera.In summary, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand. Further promising candidates are in sight for both heterologous and endogenous regulatable expression systems and future studies certainly will reveal more regulated AAB promoters. Additionally, the flexible SEVA toolkit, used within the AAB hitherto only in


Frailty is associated with an increased risk of negative short-term and long-term hospital outcomes. This study aimed to evaluate the role of frailty in predicting readmission, length of stay, and quality of life in the hospitalized older adults. This observational study was conducted at Ziaiyan Hospital, Tehran, Iran. In total, 304 participants (65–85 years), were enrolled through the inclusion criteria from August to December 2019. The frailty index (FI) was assessed by the minimum data set-home care. Readmission was obtained through telephone interviews. The length of stay was gathered by the patient’s hospital records, and the EuroQol questionnaire was used for assessing the quality of life. Data were collected by a researcher nurse at the admission time, 30, 60, and 90 days after discharge. The logistic regression model and repeated measures ANOVA were employed to analyze the association between frailty and outcomes.According to FI, 102 (33.55%) participants were pre-frail, whereas 35 (11.51%) were frail. In the fully-adjusted model for readmission, the pre-frail participants had a higher risk of readmission at the hospital in comparison with the nonfrail and frail groups , and also for GP visits, frail patients showed nearly signiﬁcant differences  but there were no differences between frail and pre-frail patients in readmissions in the emergency ward. In a fully-adjusted prolonged stay model, pre-frail patients had a higher probability to stay longer in hospital . The fully-adjusted model for QoL showed, frail patients were more prone to the declined levels of QoL in comparison with pre-frail patients .The findings indicated that frailty worsened negative outcomes and declined QoL. Early diagnosis in hospital settings could be beneficial for designing optimal care plans for the frail and pre-frail patients. With the aging population worldwide, the frailty of older adults is a concern for health systems because older patients, especially the frail older adults needing further care and services, are more likely to be hospitalized [1]. Frailty, in the hospitalized older adults, is associated with an increased risk of negative outcomes in the short term (increased length of stay and readmission) and the long term (disability and death) [2]. It could also predict loss of independence, disability, falls, delirium, re-hospitalization, and declined quality of life among the elderly [3]. Although frailty is a common problem in the hospitalized older adult patients, its diagnosis usually faces a few challenges [2]. Currently, there is no consensus for frailty assessment in clinical settings [4].Clinicians pay close attention to the impact of frailty on health aspects of life among the older adult patients. The right assessment should be applied in proper settings to diagnose frailty accurately [5]. Frailty assessment is performed based on two approaches, i.e. the phenotype model and the cumulative deficit approach. The phenotype approach measures weight loss, fatigue, exhaustion, weakness, physical activity, and mobility dysfunction [6]. Evaluation through the phenotype model can be useful for measuring the functions of the senior citizens such as gait speed and grip strength. However, it is difficult to apply this approach to the older adult patients in hospital settings that may not accurately show the baseline frailty status [4]. The other approach is the accumulation of health deficits, known as the frailty index (FI), including 30 or more deficits from different domains related to health [7]. The minimum data set (MDS) assessment form and the comprehensive geriatric assessment (CGA) allow for the measurement of the MDS-specific frailty index and CGA-FI [8]. It is hard to implement the cumulative deficit approach due to a large number of variables; however, some studies indicate that the FI is a more sensitive predictor of adverse outcomes in the older adults because of its multidimensionality [9]. In addition, the FI is a strong predictor of hospital outcomes, mortality, and disability [10]. Hence, the FI might serve as a useful approach to ascertain the effectiveness of health status in clinical settings [11].Since the MDS-HC form is used as a standard assessment instrument in hospital settings to discharge older patients, the MDS-specific frailty index can be extracted from it [12]. Therefore, the MDS-HC form can be employed to evaluate frailty and detect short-term and long-term outcomes among the hospitalized older adult patients [4]. This study aims to appraise the role of frailty in the prediction of patients outcomes  among the hospitalized older adults based on the MDS-specific frailty index.This prospective observational study was conducted on geriatric patients (n = 304) who were admitted to Ziaiyan Hospital  from August 2019 to December 2019.Since the minimum data set-home care (MDS-HC) requires accurate responses of the older adults and their caregivers, they were selected through the following criteria 1) The patients were aged between 65 and 85 years old. 2) They were admitted to geriatric, internal, and coronary care unit wards. 3). They were not admitted to ICU. 4). They were not terminal ill or in high need of care. 5). They did not reside in a nursing home. 6). The presence of a caregiver was mandatory for the consent of patients with a lack of mental capacity. Severe disease cases or the older adult who were transferred from hospitals to nursing homes were excluded, for they were unable to fill out the questionnaires or complete functional assessments. The health-related and functional variables were collected through face-to-face interviews conducted by a trained nurse at admission time based on the MDS-HC.Informed consent was obtained from patients or their legally acceptable representatives. The study was approved by the Ethics Committee of the University of Social Welfare and Rehabilitation (IR.USWR.REC.1396.296). The frailty index and other outcomes base on the following information were extracted.The MDS-HC is a standard geriatric assessment tool that contains more than 200 items regarding attention, cognition, orientation, mood and affection, function, nutrition, medication, pain, incontinence, and environment. In this study, the frailty index (FI) was constructed by using 42 health-related deficits/variables based on an FI derived from the MDS-HC. More information about the calculation of the FI was written in Burn et al. study [13]. To calculate the FI, it was necessary to answer all 42 health-related deficits/variables, so the incomplete information of the older adults was not considered in the calculation of the FI, and they were excluded from the study. Each variable was recorded on a binary scale of 0 or 1 . The FI was calculated by adding up the number of deficits recorded for a patient. The summation was then divided by the total number of possible deficits representing an FI with a potential range from 0 to 1 [13]. In this analysis, like the study by McKenzie et al., three frailty categories were obtained: nonfrail (≤0.21), pre-frail (>0.21 to ≤0.30), and frail (>0.30) [14].Readmission is defined as at least another admission to a hospital or an emergency ward or a visit to a general practitioner (GP) for any reason within 3 months after discharge [15]. Readmission information was obtained from all patients through telephone interviews conducted by a trained nurse within 30, 60, and 90 days from the baseline.The length of stay was defined as the number of days between admission and discharge (or death). The prolonged hospitalization period was also calculated for further analysis based on the following definition: “A prolonged length of stay is equal to or greater than 75% of the total length of stay in the entire cohort study” [16].The EuroQol five-dimensional questionnaire (EQ-5D-3L) was used for assessing the quality of life. This tool consists of two parts, i.e. the EQ-5D descriptive system and the EQ-5D visual analog scale (EQ-5D VAS). The EQ-5D descriptive system includes mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. The total score ranges from –0.594 to 1 based on the UK weighted index [17]. Validation of an Iranian version of “EQ-5D-3L” questionnaire has been done by Dastourani et al. study [18].In consistency with the study of Parkin et al., the results were classified as two categories in this study to define the QoL score as low quality of life (≤0.50) and high quality of life (>0.50) [19]. In the EQ-5D VAS, respondents registered the self-rated health on a vertical visual analog scale, ranging from 0  to 100  .The information of age, sex, educational attainment, marital status, co-morbidity, polypharmacy, cognition, and depression was collected to evaluate the impacts of variables that were not encoded directly in the FI. Co-morbidity is defined as the co-existence of at least 3 separate chronic illnesses [20]. Poly-pharmacy is defined as the concurrent use of more than 5 medications [21]. The patients’ cognitive states were evaluated by conducting the six-item cognitive impairment test (6-CIT) consisting of orientation, attention, and memory domains. The score ranges from 0 to 28, and scores higher than 11 indicate cognitive impairment [22].Depression was measured by the MDS-depression rating scale (MDS-DRS) with a maximum score of 14. The patient’s MDS-DRS score was interpreted based on the following category, i.e. nondepression (0), mild/moderate depression (<1 to >3), and severe depression (<3) [23]. A binary classification was used for the logistic regression. It included two categories (depression≥3 and without depression <3) [24]. Statistical analysis was performed in SPSS v.16.0  and Stata 11  at p-values <0.05 (two-tailed). The normal distribution of continuous variables was assessed by conducting the Kolmogorov–Smirnov test. The continuous and categorical variables were presented as a mean (± standard deviations) and numbers or proportions, respectively. The discrimination of frail, pre-frail, and nonfrail groups was tested through analysis of variance (ANOVA). The categorical variables were compared by using Chi-squared tests. Furthermore, the unadjusted and fully-adjusted logistic regression models were employed to estimate the odds ratio (OR) and its 95% confidence interval (CI) in frailty status as an independent variable. A repeated measure ANOVA was conducted to check the trends in quality of life over time. A total of 304 geriatric patients agreed to participate in this study; however, 16 participants were excluded due to incomplete assessment resulting from follow-up inability or death. The mean age of the older adult patients was about 75.72 ± 6.30 years. The FI maximum score was reported at 0.540, and the mean ± SD of FI scores was reported 0.21 ± 0.08. Based on FI cutoff points, 102 (33.55%) patients were identified as pre-frail, whereas 35 (11.51%) were diagnosed as frail. The frail patients were older than the pre-frail and nonfrail  patients. There were significant differences between variables  in frailty status [Table 1].Unadjusted logistic regression analysis showed significant differences between nonfrail and pre-frail patients in readmission rates at the hospital  and emergency ward . Based on results regarding frail patients compared with nonfrail and pre-frail patients, the GP visits were highly significant .After age, sex, depression, and cognition variables were adjusted, the pre-frail participants had a higher risk of readmission at the hospital in comparison with the nonfrail and frail groups . In the fully-adjusted model for the emergency ward variables, there were no significant differences between frail and pre-frail patients in readmissions. In the fully-adjusted model for GP visits, frail patients showed nearly signiﬁcant differences . In fully-adjusted logistic regression, sex  and cognitive impairment variables increased the emergency ward readmissions frequency of the elderly patients. Moreover, the pre-frail and depressed patients were more prone to GP visits in this study (Table 2).There were no significant differences between nonfrail, pre-frail, and frail geriatric patients in the length of stay. In the unadjusted logistic regression model, pre-frail  and frail patients  were more prone to prolonged stay at the hospital. In a fully-adjusted prolonged stay model, pre-frail geriatric patients had a higher probability to stay longer in hospital ; however, pre-frail and frail elderly women experienced higher levels of the length of stay in hospital [Table 2].The unadjusted logistic regression model showed significant differences between nonfrail, frail , and pre-frail elderly patients in the scores of QoL . In the fully-adjusted model, frail patients  were more prone to the declined levels of QoL in comparison with pre-frail patients. Furthermore, QoL can decline more in older frail or pre-frail patients (Table 2).The results of repeated measures ANOVA showed significant differences in QoL scores among nonfrail, pre-frail, and frail older adult patients at the baseline 30, 60, and 90 days after discharge from the hospital (p < 0.001) (Table 3). Figure shows the descending slope of QoL (EQ5D and EQ.VAS) scores at baseline, 30, 60, and 90 days after geriatric patients were discharged from the hospital.This study described that the significant differences in specific variables  concerning the frailty status of geriatric inpatients. Furthermore, a significant association was observed between frailty and prolonged stay, readmission, and QoL among the hospitalized older adult patients. The prolonged stay was prominent in pre-frail geriatric patients in the hospital. In frail geriatric patients, the probability of a GP visit was approximately significant. The QoL was declined in frail, pre-frail, and nonfrail patients during the 3-month follow-up; it decreased more in frail patients. Based on results in the present study, there were more readmissions in frail and pre-frail geriatric patients. Similarly, Vidan et al. reported that frailty was an independent predictor in the hospitalized Spanish older adults within 12 months of readmission [25]. In hospitalized Chinese patients, frailty increased the risk of readmission [26]. Additionally, in aortic valve implant older adult patients in a Japanese study, frailty was correlated with unplanned readmission [27]. The major reason for an association between frailty and readmission might be the assumption that patients admitted to hospitals were more sensitive to frailty syndrome and experienced a higher risk of readmission or poor outcomes [28]. At the same time, the discharge process might not consider the health-related concerns and needs of the older adult patients in some hospitals [29]; therefore, it may increase the chance of re-hospitalization among frail and pre-frail older patients.Results showed that cognitive impairment and sex [30] affected readmissions in frail patients. The present study also indicated higher emergency ward readmissions in men as well as cognitive impairment in frail and pre-frail patients. Existing sex differences in the findings might be attributed to health-seeking behavior and perceived health status. The majority of the older adult men were less interested in using follow-up care and preventive programs. They were also more prone to unintentional acute illnesses because of unwillingness to comply with preventive programs. This could explain the more ED readmissions in older males after discharge [31]. Possible mechanisms for increasing the probability of re-admission in cognitive impairment patients might be due to disorientation in the time or place as well as problems in complying with simple commands in the hospital as a result of attention/memory deficits [32]. Moreover, patients and caregivers are usually agitated concerning the issues that will emerge after discharge. Patients commonly fail to pay attention to the necessary instructions after discharge [33]; therefore, they are re-admitted quickly after discharge.The findings of this study showed that the probability of prolonged stay was higher in the pre-frail hospitalized older adults. This finding is consistent with the results of other studies . Apparently, the Iranian pre-frail older adult patients were more prone to lengthy hospital stay [35]. This might be because the frail patients were mostly bed-ridden in their homes [35] due to being mistreated by their family caregivers or facing ageism taboo, which might have been neglected by their family caregivers [36]. The readmission rate was lower in the elderly frail patients than in the pre-frail patients.According to the findings, the older adult women were more likely to stay in hospital. This was consistent with the findings reported by De Buyser et al. [37]. However, Alnajashia et al. [38] found no significant association between length of stay and sex. It might be due to higher levels of life expectancy in the older adult women, compared with older men, as well as the high probability to live alone and the high rate of co-morbidity in the Iranian older female than the male older adults [39]. Besides, there is no social security system in Iran to support the elderly , especially for older women. As a result, the older women may stay longer in hospital.Based on the research findings, the older adult frail patients had a lower QoL score. In a similar study, Cavrini et al. reported that the QoL score was correlated with the number of hospitalization and institutionalization in the Italian older adults within two years of follow-up [40]. Kahlon et al. observed that frail patients had lower QoL scores than nonfrail older adult patients in Canada [41]. In contrast, Kojima et al. noted that the British pre-frail older adult patients not only had a better QoL score at baseline but also showed improvements in QoL over time [42]. However, the research settings of our study are not similar to those of the reviewed studies.In this study, the reason for a lower score of QoL in frail older adult patients might be interpreted as the fact that hospitalization reduced the mobility and functional capacity of the older adults and increased dependency [43]. Meanwhile, independence, and self-care are important measures in the lives of the older adults which are disrupted during hospitalization; thus, it appears that hospitalization decreased QoL in frail patients.There were a few research limitations. This is a single-center study, the findings of which might not be generalizable. Only one frailty assessment tool (MDS-HC frailty index) was employed due to its practicality, ease of administration, and complete assessment of multiple important geriatric domains. The cause and duration of each readmission were not discussed in the study evaluation intervals.In the present study, the frailty assessment was performed using MDS-HC in the hospital setting, since adopting frailty measures depends greatly on clinical settings and the purpose of frailty assessment [26]. The results obtained revealed that the MDS-specified frailty index was able to predict the adverse outcomes in the hospitalized older adult patients. Based on the MDS-specified frailty index, the pre-frail status was more prevalent among geriatric inpatients. This is a valuable finding for policymakers so that they can be aware of the vulnerable older adult population in Iran and prepare appropriate care plans for major inpatient vulnerable groups. Informed consent was obtained from all patients or their legally acceptable representatives. The study was approved by the Ethics Committee of the University of Social Welfare and Rehabilitation(IR.USWR.REC.1396.296).