Environmental Aspects of Zoonotic Diseases

Environmental Aspects of Zoonotic Diseases provides a definitive description, commentary and research needs of environmental aspects related to zoonotic diseases. There are many interrelated connections between the environment and zoonotic diseases such as: water, soil, air and agriculture. The book presents investigations of these connections, with specific reference to environmental processes such as: deforestation, floods, draughts, irrigation practices, soil transfer and their impact on bacterial, viral, fungal, and parasitological spread. Environmental aspects such as climate (tropical, sub-tropical, temperate, arid and semi-arid), developed and undeveloped countries, animal traffic animal border crossing, commercial animal trade, transportation, as well geography and weather on zoonosis, are also discussed and relevant scientific data is condensed and organized in order to give a better picture of interrelationship between the environment and current spread of zoonotic diseases

Performance comparison of plant root biofilm, gravel attached biofilm and planktonic microbial populations, in phenol removal within a constructed wetland wastewater treatment system

This study was performed in order to understand the relative contribution of a constructed wetland (CW) system’s various components to phenol degradation (100 mg∙L–1) under controlled plant biomass/gravel/ water experimental ratios. This was done by division of a pilot-scale CW system into its components, with or without their associated bacteria: (i) gravel, plant and water; (ii) gravel and water; (iii) water; (iv) gravel; (v) plant; (vi) control (sterile water). The highest phenol biodegradation rate occurred for the gravel-attached biofilm followed by root-attached biofilm and planktonic population, which recorded a similar rate to each other and a much lower rate than the gravel-attached biofilm. A control containing CW planktonic inactivated bacteria (autoclaved water) did not impact phenol removal, revealing that microbial populations are the major factor in phenol removal. The differences in the phenol removal achieved could be attributed to higher numbers of specific phenol degraders on the gravel surface, compared to lower numbers of root-attached and planktonic bacterial fractions, as isolated using phenol-agar plates which contained phenol as the sole carbon source. The main contributor to our findings appears to be the larger surface area provided by the gravel bed compared to plant roots.Keywords: biofilm; constructed wetland; gravel; microbial activity; pheno

Live-cell protein labelling with nanometre precision by cell squeezing

Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (~1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy

Whole Cell Imprinting in Sol-Gel Thin Films for Bacterial Recognition in Liquids: Macromolecular Fingerprinting

Thin films of organically modified silica (ORMOSILS) produced by a sol-gel method were imprinted with whole cells of a variety of microorganisms in order to develop an easy and specific probe to concentrate and specifically identify these microorganisms in liquids (e.g., water). Microorganisms with various morphology and outer surface components were imprinted into thin sol-gel films. Adsorption of target microorganism onto imprinted films was facilitated by these macromolecular fingerprints as revealed by various microscopical examinations (SEM, AFM, HSEM and CLSM). The imprinted films showed high selectivity toward each of test microorganisms with high adsorption affinity making them excellent candidates for rapid detection of microorganisms from liquids

In vitro and ex vivo strategies for intracellular delivery

Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.National Institutes of Health (U.S.) (R01GM101420-01A1

Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform

Intracellular delivery of materials is a challenge in research and therapeutic applications. Physical methods of plasma membrane disruption have recently emerged as an approach to facilitate the delivery of a variety of macromolecules to a range of cell types. We use the microfluidic CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content of the surrounding buffer (recovery time ~5 min without calcium vs. ~30 s with calcium). Moreover, we illustrate that manipulation of the membrane repair kinetics can yield up to 5× improvement in delivery efficiency without significantly impacting cell viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells. Subsequent manipulation of membrane repair kinetics also enables the delivery of larger materials, such as antibodies, to these difficult to manipulate cells. This work provides insight into the membrane repair process in response to mechanical delivery and could potentially enable the development of improved delivery methods.National Institutes of Health (U.S.) (Grant RC1 EB011187-02)National Institutes of Health (U.S.) (Grant R01GN101420-01A1)Kathy and Curt Marble Cancer Research FundNational Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant MPP-09Call-Langer-60

Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8[superscript +]T-cells, and not CD4[superscript +]T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8[superscript +]T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8[superscript +]T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8[superscript +]T-cells, and decoupling of antigen uptake from B-cell activation.Kathy and Curt Marble Cancer Research Fund (Frontier Research Programme Grant)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32CA180586

Extrapulmonary tuberculosis in HIV-infected patients in rural Tanzania: the prospective Kilombero and Ulanga antiretroviral cohort

In sub-Saharan Africa, diagnosis and management of extrapulmonary tuberculosis (EPTB) in people living with HIV (PLHIV) remains a major challenge. This study aimed to characterize the epidemiology and risk factors for poor outcome of extrapulmonary tuberculosis in people living with HIV (PLHIV) in a rural setting in Tanzania.; We included PLHIV >18 years of age enrolled into the Kilombero and Ulanga antiretroviral cohort (KIULARCO) from 2013 to 2017. We assessed the diagnosis of tuberculosis by integrating prospectively collected clinical and microbiological data. We calculated prevalence- and incidence rates and used Cox regression analysis to evaluate the association of risk factors in extrapulmonary tuberculosis (EPTB) with a combined endpoint of lost to follow-up (LTFU) and death.; We included 3,129 subjects (64.5% female) with a median age of 38 years (interquartile range [IQR] 31-46) and a median CD4+ cell count of 229/μl (IQR 94-421) at baseline. During the median follow-up of 1.25 years (IQR 0.46-2.85), 574 (18.4%) subjects were diagnosed with tuberculosis, whereof 175 (30.5%) had an extrapulmonary manifestation. Microbiological evidence by Acid-Fast-Bacillus stain (AFB-stain) or Xpert® MTB/RIF was present in 178/483 (36.9%) patients with pulmonary and in 28/175 (16.0%) of patients with extrapulmonary manifestations, respectively. Incidence density rates for pulmonary Tuberculosis (PTB and EPTB were 17.9/1000person-years (py) (95% CI 14.2-22.6) and 5.8/1000 py (95% CI 4.0-8.5), respectively. The combined endpoint of death and LTFU was observed in 1058 (33.8%) patients, most frequently in the subgroup of EPTB (47.2%). Patients with EPTB had a higher rate of the composite outcome of death/LTFU after TB diagnosis than with PTB [HR 1.63, (1.14-2.31); p = 0.006]. The adjusted hazard ratios [HR (95% CI)] for death/LTFU in EPTB patients were significantly increased for patients aged >45 years [HR 1.95, (1.15-3.3); p = 0.013], whereas ART use was protective [HR 0.15, (0.08-0.27); p <0.001].; Extrapulmonary tuberculosis was a frequent manifestation in this cohort of PLHIV. The diagnosis of EPTB in the absence of histopathology and mycobacterial culture remains challenging even with availability of Xpert® MTB/RIF. Patients with EPTB had increased rates of mortality and LTFU despite early recognition of the disease after enrollment