In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.

BackgroundPhysical inactivity is a growing public health problem, and the fourth leading risk factor for global mortality. Conversely, indigenous populations living traditional lifestyles reportedly engage in vigorous daily activity that is protective against non-communicable diseases. Here we analyze physical activity patterns among the Tsimane, forager-horticulturalists of Amazonian Bolivia with minimal heart disease and diabetes. We assess age patterns of adult activity among men and women, test whether modernization affects activity levels, and examine whether nascent obesity is associated with reduced activity.Methods and FindingsA factorial method based on a large sample of behavioral observations was employed to estimate effects of age, sex, body mass index, and modernization variables on physical activity ratio (PAR), the ratio of total energy expenditure to basal metabolic rate. Accelerometry combined with heart rate monitoring was compared to the factorial method and used for nighttime sampling. Tsimane men and women display 24 hr physical activity level (PAL) of 2.02–2.15 and 1.73–1.85, respectively. Little time was spent “sedentary”, whereas most activity was light to moderate, rather than vigorous. Activity peaks by the late twenties in men, and declines thereafter, but remains constant among women after the early teens. Neither BMI, fat free mass or body fat percentage are associated with PAR. There was no negative effect of modernization on physical activity.ConclusionsTsimane display relatively high PALs typical of other subsistence populations, but of moderate intensity, and not outside the range of developed populations. Despite rapidly increasing socioeconomic change, there is little evidence that total activity has yet been affected. Overweight and obesity are more prevalent among women than men, and Spanish fluency is associated with greater obesity in women. The lack of cardiovascular disease among Tsimane is unlikely caused by activity alone; further study of diet, food intake and infectious disease is needed.

Objectives Resting metabolic rate (RMR) reflects energetic costs of homeostasis and accounts for 60-75% of total energy expenditure (TEE). Lean mass and physical activity account for much RMR variability, but the impact of prolonged immune activation from infection on human RMR is unclear in naturalistic settings. We evaluate the effects of infection on mass-corrected RMR among Bolivian forager-horticulturalists, and assess whether RMR declines more slowly with age than in hygienic sedentary populations, as might be expected if older adults experience high pathogen burden. Materials and Methods RMR was measured by indirect calorimetry (Fitmate MED, Cosmed) in 1,300 adults aged 20-90 and TEE was measured using doubly labeled water (n= 40). Immune biomarkers, clinical diagnoses and anthropometrics were collected by the Tsimane Health and Life History Project. Results Tsimane have higher RMR and TEE than people in sedentary industrialized populations. Tsimane RMR is 18-47% (women) and 22-40% (men) higher than expected using six standard prediction equations. Tsimane mass-corrected TEE is similarly elevated compared to Westerners. Elevated leukocytes and helminths are associated with excess RMR in multivariate regressions, and jointly result in a predicted excess RMR of 10-15%. After age 40, RMR declines by 69 kcal/decade (p<0.0001). Controlling for lean mass and height accounts for 71% of age-related RMR decline, and adding indicators of infection minimally affects the age slope. The residual level of age-related decline from age 40 is 1.2% per decade. Conclusion High pathogen burden may lead to higher metabolic costs, which may be offset by smaller body mass or other energy-sparing mechanisms.

The challenge hypothesis posits that acute increases in testosterone (T) during male-male competition enhance performance and survivability while limiting the physiological costs of consistently high T. Human challenge hypothesis research focuses on young men in industrial populations, who have higher baseline T levels than men in subsistence populations. We tested whether the Tsimane, pathogenically stressed forager-horticulturalists of the Bolivian Amazon, would express acute T increases in response to physical competition. Saliva was collected from 88 Tsimane men (aged 16-59 years) before and after a competitive soccer match. Tsimane men had significantly lower baseline levels of T (b ¼ 20.41, p , 0.001) when compared with age-matched United States (US) males. Linear mixed-effects models were used to establish that T increased significantly immediately following competition (b ¼ 0.23, p , 0.001), remaining high 1 h later (b ¼ 0.09, p ¼ 0.007); equivalent to 30.1 and 15.5 per cent increases in T, respectively. We did not find larger increases in T among winners (p ¼ 0.412), although T increases were positively associated with self-rated performance (b ¼ 9.07, p ¼ 0.004). These results suggest that despite lower levels of T than US males, Tsimane males exhibit acute increases in Tat the same relative magnitude reported by studies in industrialized settings, with larger increases in T for those who report better individual performance.

Introduction:We evaluated the prevalence of dementia and mild cognitive impairment (MCI) in indigenous Tsimane and Moseten, who lead a subsistence lifestyle. Methods: Participants from population-based samples ≥ 60 years of age (n = 623) were assessed using adapted versions of the Modified Mini-Mental State Examination, informant interview, longitudinal cognitive testing and brain computed tomography (CT) scans. Results: Tsimane exhibited five cases of dementia (among n = 435; crude prevalence = 1.2%, 95% confidence interval [CI]: 0.4, 2.7); Moseten exhibited one case (among n = 169; crude prevalence = 0.6%, 95% CI: 0.0, 3.2), all age ≥ 80 years.Age-standardized MCI prevalence was 7.7% (95% CI: 5.2, 10.3) in Tsimane and 9.8% (95% CI: 4.9, 14.6) in Moseten. Cognitive impairment was associated with visuospatial impairments, parkinsonian symptoms, and vascular calcification in the basal ganglia. Discussion:The prevalence of dementia in this cohort is among the lowest in the world.Widespread intracranial medial arterial calcifications suggest a previously unrecognized, non-Alzheimer's disease (AD) dementia phenotype.