Changes of composition and antibiotic resistance of fecal coliform bacteria in municipal wastewater treatment plant.

The dynamics of the composition and antibiotic resistance of the fecal coliform bacteria (FCB) in a typical wastewater treatment plant (WWTP) were investigated concerning the seasonal changes. Results showed that WWTP could remove the FCB concentration by 3∼5 logs within the effluent of 104∼105 CFU/L, but the antibiotic resistant rate of FCB species increased significantly after WWTP. The dominant FCB changed from Escherichia coli in the influent (∼73.0%) to Klebsiella pneumoniae in the effluent (∼53.3%) after WWTP, where the Escherichia coli was removed the most, while Klebsiella pneumoniae was the most persistent. The secondary tank removed the most of FCB (by 3∼4 logs) compared to other processes, but increased all the concerned antibiotic resistant rate. The potential super bugs of FCB community showing resistance to all the target antibiotics were selected in the biological treatment unit of WWTP. The FCB showed the highest multiple antibiotic resistance (92.9%) in total which even increased to 100% in the effluent. Klebsiella has the highest antibiotic resistant rate in FCB, with a multiple antibiotic resistance rate of 98.4%. These indicated that the Klebsiella pneumoniae not just Escherichia coli should be specially emphasized after WWTP concerning the health risk associated with FCB community.

Synaptic plasticity in human thalamocortical assembloids.

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.

Synaptic plasticity in human thalamocortical assembloids.

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.

Heterogeneity of cancer-associated fibroblasts in head and neck squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) consist of heterogeneous cellular populations that contribute critical roles in head and neck squamous cell carcinoma (HNSCC). A series of computer-aided analyses were performed to determine various aspects of CAFs in HNSCC, including their cellular heterogeneity, prognostic value, relationship with immune suppression and immunotherapeutic response, intercellular communication, and metabolic activity. The prognostic significance of CKS2+ CAFs was verified using immunohistochemistry. Our findings revealed that fibroblasts group demonstrated prognostic significance, with the CKS2+ subset of inflammatory CAFs (iCAFs) exhibiting a significant correlation with unfavorable prognosis and being localized in close proximity to cancer cells. Patients with a high infiltration of CKS2+ CAFs had a poor overall survival rate. There is a negative correlation between CKS2+ iCAFs and cytotoxic CD8+ T cells and natural killer (NK) cells, while a positive correlation was found with exhausted CD8+ T cells. Additionally, patients in Cluster 3, characterized by a high proportion of CKS2+ iCAFs, and patients in Cluster 2, characterized by a high proportion of CKS2- iCAFs and CENPF-/MYLPF- myofibroblastic CAFs (myCAFs), did not exhibit significant immunotherapeutic responses. Moreover, close interactions was confirmed to exist between cancer cells and CKS2+ iCAFs/ CENPF+ myCAFs. Furthermore, CKS2+ iCAFs demonstrated the highest level of metabolic activity. In summary, our study enhances the understanding of the heterogeneity of CAFs and provided insights into improving the efficacy of immunotherapies and prognostic accuracy for HNSCC patients.

Improving the efficiency of anaerobic digestion of Molasses alcohol wastewater using Cassava alcohol wastewater as a mixed feedstock.

Cassava alcohol wastewater (CAW) was utilized as a mixed feedstock to explore whether the addition of CAW could improve the anaerobic digestion of Molasses alcohol wastewater (MAW). The result showed that the rate of removal of the soluble chemical oxygen demand in the M treatment mixed with CAW was 70.13 ± 0.16%, which was significantly higher than that of the C treatment (only MAW), which was 61.23 ± 0.36%. Co-digestion in the M treatment resulted in higher methane production, achieving 23.89% increase in methane yield compared to C treatment. The addition of CAW helps to alleviate the accumulation of volatile fatty acids (397.06 ± 141.82 mg·L-1), enhance the stability of system and promote the establishment of stable and active microbial communities. Microbial community structure analysis indicated that hydrolytic bacteria such as Bacteroidetes, Firmicutes, and Proteobacteria, and acetoclastic methanogens, including Methanosaeta and Methanosarcina were more abundant in the co-digests.

A Case for Thalamic Mechanisms of Schizophrenia: Perspective From Modeling 22q11.2 Deletion Syndrome.

Schizophrenia is a severe, chronic psychiatric disorder that devastates the lives of millions of people worldwide. The disease is characterized by a constellation of symptoms, ranging from cognitive deficits, to social withdrawal, to hallucinations. Despite decades of research, our understanding of the neurobiology of the disease, specifically the neural circuits underlying schizophrenia symptoms, is still in the early stages. Consequently, the development of therapies continues to be stagnant, and overall prognosis is poor. The main obstacle to improving the treatment of schizophrenia is its multicausal, polygenic etiology, which is difficult to model. Clinical observations and the emergence of preclinical models of rare but well-defined genomic lesions that confer substantial risk of schizophrenia (e.g., 22q11.2 microdeletion) have highlighted the role of the thalamus in the disease. Here we review the literature on the molecular, cellular, and circuitry findings in schizophrenia and discuss the leading theories in the field, which point to abnormalities within the thalamus as potential pathogenic mechanisms of schizophrenia. We posit that synaptic dysfunction and oscillatory abnormalities in neural circuits involving projections from and within the thalamus, with a focus on the thalamocortical circuits, may underlie the psychotic (and possibly other) symptoms of schizophrenia.

Investigation of the Kinetics and Reaction Mechanism for Photodegradation Tetracycline Antibiotics over Sulfur-Doped Bi2WO6-x/ZnIn2S4 Direct Z-Scheme Heterojunction.

The rational design of direct Z-scheme heterostructural photocatalysts using solar energy is promising for energy conversion and environmental remediation, which depends on the precise regulation of redox active sites, rapid spatial separation and transport of photoexcited charge and a broad visible light response. The Bi2WO6 materials have been paid more and more attention because of their unique photochemical properties. In this study, S2- doped Bi2WO6-x coupled with twin crystal ZnIn2S4 nanosheets (Sov-BWO/T-ZIS) were prepared as an efficient photocatalyst by a simple hydrothermal method for the removal of tetracycline hydrochloride (TCH). Multiple methods (XRD, TEM, XPS, EPR, UV vis DRS, PL etc.) were employed to systematically investigate the morphology, structure, composition and photochemical properties of the as-prepared samples. The XRD spectrum indicated that the S2- ions were successfully doped into the Sov-BWO component. XPS spectra and photoelectrochemical analysis proved that S2- served as electronic bridge and promoted captured electrons of surface oxygen vacancies transfer to the valence band of T-ZIS. Through both experimental and in situ electron paramagnetic resonance (in situ EPR) characterizations, a defined direct Z-scheme heterojunction in S-BWO/T-ZIS was confirmed. The improved photocatalytic capability of S-BWO/T-ZIS results ascribed that broadened wavelength range of light absorption, rapid separation and interfacial transport of photoexcited charge, precisely regulated redox centers by optimizing the interfacial transport mode. Particularly, the Sov-50BWO/T-ZIS Z-scheme heterojunction exhibited the highest photodegradation rate was 95% under visible light irradiation. Moreover, this heterojunction exhibited a robust adsorption and degradation capacity, providing a promising photocatalyst for an organic pollutant synergistic removal strategy.

Association between Plasma Urotensin II and Risk of Hypertension: Findings from a Prospective Study.

Up to date, human urotensin II (UII) is the most potent vasoconstrictor in mammalian animals. To explore whether UII played an important role in the development of hypertension, we conducted a prospective study in Changshu city, China. The baseline investigation was carried out in 2007, and the first follow-up investigation was conducted in 2013. From the participants, we randomly obtained 2000 normotensive subjects aged 40 years and older without any severe disease at baseline and examined plasma UII and endothelin-1 (ET-1) with their blood samples at baseline. Logistic models were used to analyze the association between baseline UII, baseline ET-1, and newly occurring hypertension. In 1,819 subjects with complete data, 723 subjects developed into hypertensive in about five years. After adjusting some potential confounders, the odds ratio (95% confidence interval) for risk of hypertension comparing the highest with the lowest quartile of baseline UII was 0.888 (0.689-1.144). The role of UII in the development of hypertension was not found in the current study; therefore, further research studies should be conducted to explore the relationship between UII and hypertension.

Microalbuminuria mediates the association between serum uric acid and elevation of blood pressure: a longitudinal analysis in the Gusu cohort.

OBJECTIVES: Although hyperuricemia, microalbuminuria, and hypertension are highly correlated, their temporal relationship is largely unknown. We aimed to examine whether microalbuminuria mediated the association between hyperuricemia and hypertension. METHODS: Leveraging a longitudinal cohort including 1981 Chinese adults who had blood pressures, urinary albumin to creatinine ratio (UACR), and uric acid measured twice 4 years apart, we examined the temporal relationships among hyperuricemia, microalbuminuria, and hypertension by cross-lagged panel analysis followed by a causal mediation analysis to confirm the temporal consequence. Age, sex, education level, cigarette smoking, alcohol consumption, obesity, blood glucose, and lipids were adjusted. RESULTS: The cross-lagged panel analysis demonstrated that the relationship from baseline UACR to follow-up uric acid was significantly smaller than that from baseline uric acid to follow-up UACR (ß: 0.010 vs. 0.054, P < 0.001). The relationships from baseline blood pressures to follow-up UACR were also significantly smaller than that from baseline UACR to follow-up blood pressures (ß: 0.031 vs. 0.092, P < 0.001 for systolic and ß: 0.015 vs. 0.096, P < 0.001 for diastolic). The causal mediation analysis found that UACR partially mediated the association of baseline uric acid with follow-up SBP (mediate proportion: 9.14%, 95% CI: 1.58-23.00%) and DBP (mediate proportion: 7.38%, 95% CI: 1.05-19.00%). CONCLUSION: Microalbuminuria may follow elevated uric acid and partially mediate its effect on future risk of hypertension in Chinese adults.

Bidirectional and Temporal Association Between Hypertension and Microalbuminuria: A Longitudinal Study in Chinese Adults.

Background Although hypertension and microalbuminuria are closely interrelated, the magnitude and temporal sequence of the bidirectional association between hypertension and microalbuminuria are largely unknown. We aimed to delineate the bidirectional and temporal relationship between hypertension and microalbuminuria. Methods and Results Leveraging a longitudinal cohort of Chinese adults who had blood pressure and urinary albumin measured twice 4 years apart, we examined the temporal association between hypertension and microalbuminuria by bidirectional and cross-lagged panel analysis. All participants were free of cardiovascular disease and chronic kidney disease at baseline. Bidirectional association analysis found that baseline microalbuminuria predicted the risk of incident hypertension (odds ratio=1.75, P=0.028), and baseline blood pressure also significantly predicted the risk of microalbuminuria (odds ratios=1.27 and 1.21 for a per-SD increase in systolic and diastolic blood pressure, respectively; all P<0.05). Cross-lagged panel analysis demonstrated a bottom-line significant relationship of baseline systolic blood pressure to follow-up urinary albumin ( P=0.079), which is significantly weaker than the other direction of the relationship of baseline urinary albumin to follow-up blood pressures (all P<0.001). Conclusions These findings indicate a significant bidirectional association between microalbuminuria and hypertension in Chinese adults. Elevated urinary albumin excretion is more likely to precede hypertension. The causality between microalbuminuria and hypertension needs further investigation.

Activation of axon initial segmental GABAA receptors inhibits action potential generation in neocortical GABAergic interneurons.

Ionotropic GABAA receptors expressing at the axon initial segment (AIS) of glutamatergic pyramidal cell (PC) in the cortex plays critical roles in regulating action potential generation. However, it remains unclear whether these receptors also express at the AIS of cortical GABAergic interneurons. In mouse prefrontal cortical slices, we performed experiments at the soma and AIS of the two most abundant GABAergic interneurons: parvalbumin (PV) and somatostatin (SST) positive neurons. Local application of GABA at the perisomatic axonal regions could evoke picrotoxin-sensitive currents with a reversal potential near the Cl- equilibrium potential. Puffing agonists to outside-out patches excised from AIS confirmed the expression of GABAA receptors. Further pharmacological experiments revealed that GABAA receptors in AIS of PV neurons contain α1 subunits, different from those containing α2/3 in AIS and α4 in axon trunk of layer-5 PCs. Cell-attached recording at the soma of PV and SST neurons revealed that the activation of AIS GABAA receptors inhibits the action potential generation induced by synaptic stimulation. Together, our results demonstrate that the AIS of PV and SST neurons express GABAA receptors with distinct subunit composition, which exert an inhibitory effect on neuronal excitability in these inhibitory interneurons.

Firing Frequency Maxima of Fast-Spiking Neurons in Human, Monkey, and Mouse Neocortex.

Cortical fast-spiking (FS) neurons generate high-frequency action potentials (APs) without apparent frequency accommodation, thus providing fast and precise inhibition. However, the maximal firing frequency that they can reach, particularly in primate neocortex, remains unclear. Here, by recording in human, monkey, and mouse neocortical slices, we revealed that FS neurons in human association cortices (mostly temporal) could generate APs at a maximal mean frequency (Fmean) of 338 Hz and a maximal instantaneous frequency (Finst) of 453 Hz, and they increase with age. The maximal firing frequency of FS neurons in the association cortices (frontal and temporal) of monkey was even higher (Fmean 450 Hz, Finst 611 Hz), whereas in the association cortex (entorhinal) of mouse it was much lower (Fmean 215 Hz, Finst 342 Hz). Moreover, FS neurons in mouse primary visual cortex (V1) could fire at higher frequencies (Fmean 415 Hz, Finst 582 Hz) than those in association cortex. We further validated our in vitro data by examining spikes of putative FS neurons in behaving monkey and mouse. Together, our results demonstrate that the maximal firing frequency of FS neurons varies between species and cortical areas.

A Subtype of Inhibitory Interneuron with Intrinsic Persistent Activity in Human and Monkey Neocortex.

A critical step in understanding the neural basis of human cognitive functions is to identify neuronal types in the neocortex. In this study, we performed whole-cell recording from human cortical slices and found a distinct subpopulation of neurons with intrinsic persistent activity that could be triggered by single action potentials (APs) but terminated by bursts of APs. This persistent activity was associated with a depolarizing plateau potential induced by the activation of a persistent Na+ current. Single-cell RT-PCR revealed that these neurons were inhibitory interneurons. This type of neuron was found in different cortical regions, including temporal, frontal, occipital, and parietal cortices in human and also in frontal and temporal lobes of nonhuman primate but not in rat cortical tissues, suggesting that it could be unique to primates. The characteristic persistent activity in these inhibitory interneurons may contribute to the regulation of pyramidal cell activity and participate in cortical processing.

KIF5B promotes the forward transport and axonal function of the voltage-gated sodium channel Nav1.8.

Nav1.8 is a tetrodotoxin-resistant voltage-gated sodium channel selectively expressed in primary sensory neurons. Peripheral inflammation and nerve injury induce Nav1.8 accumulation in peripheral nerves. However, the mechanisms and related significance of channel accumulation in nerves remains unclear. Here we report that KIF5B promotes the forward transport of Nav1.8 to the plasma membrane and axons in dorsal root ganglion (DRG) neurons of the rat. In peripheral inflammation induced through the intraplantar injection of complete Freund's adjuvant, increased KIF5 and Nav1.8 accumulation were observed in the sciatic nerve. The knock-down of KIF5B, a highly expressed member of the KIF5 family in DRGs, reduced the current density of Nav1.8 in both cultured DRG neurons and ND7-23 cells. Overexpression of KIF5B in ND7-23 cells increased the current density and surface expression of Nav1.8, which were abolished through brefeldin A treatment, whereas the increases were lost in KIF5B mutants defective in ATP hydrolysis or cargo binding. Overexpression of KIF5B also decreased the proteasome-associated degradation of Nav1.8. In addition, coimmunoprecipitation experiments showed interactions between the N terminus of Nav1.8 and the 511-620 aa sequence in the stalk domain of KIF5B. Furthermore, KIF5B increased Nav1.8 accumulation, Nav1.8 current, and neuronal excitability detected in the axons of cultured DRG neurons, which were completely abolished by the disruption of interactions between KIF5B and the N terminus of Nav1.8. Therefore, our results reveal that KIF5B is required for the forward transport and axonal function of Nav1.8, suggesting a mechanism for axonal accumulation of Nav1.8 in inflammatory pain.

[Self-assembly and in vitro and in vivo evaluation of spherical crystallized interferon for sustained delivery].

It is a challenging and important project to prolong the in vivo half life of protein and peptide drugs by physicochemical methods without new molecular entities generation. Protein crystallization provides a new strategy for improving the stability and in vivo delivery of these drugs. We show here that recombinant human interferon-alpha (rhIFN) can form spherical crystals. The physical and chemical features of the crystals were characterized, and drug dissolution was determined in vitro. The pharmacokinetics of crystallized interferon after sc injection in rabbit at 1.5 x 10(7) U x kg(-1) was compared to that of soluble form. The crystals were characterized as mono-dispersed spheres, with yield of > 80%, mean diameter size of about 16 microm and crystallinity of 23.2%. The in vitro dissolution behavior of crystallized rhIFN was featured as low initial burst release (21% within the first 2 h) and prolonged cumulative dissolution time up to 72 h without biological potency lost. After sc administration of soluble and crystallized interferon in rabbits, the peak time (T(max)) and half life (t1/2) were prolonged from (1.80 +/- 0.45) h and (1.35 +/- 0.35) h to (13.20 +/- 2.68) h and (10.68 +/- 1.97) h, respectively. The corresponding peak concentration decreased from (1 411.10 +/- 575.28) U x mL(-1) to (721.37 +/- 206.55) U x mL(-1). PK/PD analysis indicated that (96.87 +/- 20.30) % of relative bioavailability was obtained. The research results of this work will provide important academic value and application prospect for improving clinical therapeutic effect and development of biomacromolecules delivery system for protein and peptide drugs.

Metal ions guided self-assembly of therapeutic proteins for controllable release: from random to ordered aggregation.

PURPOSE: To make a comparative study on sustained delivery performance of rhIFN with random amorphous and spherical crystal-like ordered self-assemblies. METHODS: The rhIFN self-assemblies were identified in batch crystallization mode. Physico-chemical characteristics were compared, including morphology, XRD, FTIR, CD, biological potency, the dissolution behaviors in vitro and plasma pharmacokinetics in vivo. Moreover, molecular simulation was performed to better understand their binding site and mode. RESULTS: Here, we suggest that random amorphous and spherical ordered self-assemblies allow for long action without new molecular entities generation or carriers employed. By manipulating supersaturation, the ordered aggregates were self-organized at high concentration of Zn(II) (>100 mM) in pH 5.5-6.0, which was the first time that spherical semi-crystals of rhIFN can act as a depot source for the sustained delivery of biologically active proteins. The secondary structure and biological potency of rhIFN were unchanged after aggregation. Compared with that of the native rhIFN, both self-assemblies exhibited slower absorption and extended elimination profiles after s.c. administration, which were characterized as 4.75 ± 0.82 h and 10.58 ± 1.86 h of terminal half-life for random amorphous and spherical ordered self-assemblies, respectively. CONCLUSIONS: The work described here demonstrates the possibility of self-assemblies of biomacromolecules for controllable release application of therapeutic proteins.

Polycationic peptide guided spherical ordered self-assembly of biomacromolecules.

Achieving effective controllable delivery of therapeutic biomacromolecules for long action without new molecular entities generation or carriers employed offers a promising alternative and significant clinical benefit. We show here that recombinant human interferon-alpha (rhIFN) can form a three dimensional ordered structure that is featured by spherical semi-crystalline through molecular self-assembly directed by a polycationic short peptide. The phase diagrams for self-assembly were constructed to identify the optimal regions for nucleation and ordered growth, and which were followed by the physico-chemical characterization of the ordered self-assemblies, including morphology, particle size, X-ray diffraction, circular dichroism and biological potency evaluations. With varied molar ratio of the two composed biomacromolecules, the dissolution behaviors of the self-assemblies could be manipulated in vitro and in vivo. The plasma pharmacokinetics suggested that s.c. administration of self-assemblies at the specified relative proportion of rhIFN to polycationic peptide offered a significant prolonged duration time of rhIFN blood levels up to seven days. Moreover, molecular simulation was performed to better understand their binding site and mode. The work described here demonstrates the possibility of spherical ordered self-assembly of biomacromolecules for controllable delivery application of therapeutic proteins.

Polymer-mediated anti-solvent crystallization of nitrendipine: monodispersed spherical crystals and growth mechanism.

PURPOSE: To investigate anti-solvent crystallization and growth mechanism of nitrendipine spherical crystals in an aqueous solution containing polymeric additives. METHODS: Size and shape of crystals were investigated using laser diffractometry, optical microscopy and scanning electron microscopy (SEM). Crystalline form was determined by X-ray powder diffractometer (XRPD). During crystal growth, morphological changes at different time points were observed using SEM. RESULTS: Morphology of nitrendipine crystals was affected by polymers and temperature. Monodispersed micro-spherical crystals were obtained when polyvinyl alcohol (PVA) and PEG 200 were present in crystallization medium at 2°C. During crystallization, large number of amorphous nanoparticles was first observed, followed by aggregation into a core for spherical crystals. Once crystalline state was achieved, rapid growth on core surface was observed with amorphous particles acting as a reservoir allowing formation of star-like particles with needle-like subunits. Spherical crystals were formed by filling the gap between needle-like distinct crystalline units of star-like templates with molecules from dissolved amorphous particles. CONCLUSIONS: Monodispersed nitrendipine spherical crystals were obtained using carefully controlled conditions. A mechanism for the nitrendipine spherical crystal growth is suggested. These findings provide a new insight into spherulitic crystallization of active pharmaceutical ingredients.

Protein spherulites for sustained release of interferon: preparation, characterization and in vivo evaluation.

This study develops protein spherulite, a new form of protein aggregation, for sustained-release applications of recombinant human interferon α-2b (rhIFN). rhIFN spherulites were prepared with different pH solutions with different kinds and concentrations of zinc salts at various incubation temperatures. rhIFN spherulites produced under different systems were of different morphology properties and in vitro release characters. Size-exclusion high-performance liquid chromatography analysis has shown that no protein aggregates were generated during spherulite formation, and cytopathic inhibition assay has demonstrated that the spherulitic rhIFN well maintained its structure and antiviral activity. In vivo studies showed that rhIFN spherulites provided a significantly prolonged pharmacokinetics profile profile as compared with the soluble rhIFN formulation, and the relative bioavailability based on serum rhIFN levels was about 170%. The work described here demonstrates the possibility of protein spherulites as a long-acting formulation for rhIFN.

Protamine modified metal ion-protein chelate microparticles for sustained release of interferon.

This study focuses on extending the release period of zinc-protein chelate through protamine modification. Recombinant human interferon-α-2b (rhIFN), a highly pleiotropic cytokine with a short intrinsic pharmacokinetic half-life when injected subcutaneously (∼2-6 h), was used as a model drug. Protamine modified zinc-rhIFN chelate microparticles were prepared by co-precipitating rhIFN with zinc and protamine. Introduction of protamine (2.5-20 mg/mL) into the chelation system had several prominent effects. First, percentage of chelated rhIFN was lowered (from >99% to ∼90%); second, particle size was gradually increased (from ∼0.45 µm to ∼2 µm); last but important, it extended the release period of the chelate both in vitro (complete release was retarded from 8 h to 48 h) and in vivo (t(1/2) was prolonged from 4.5 h to 15.5 h and mean residence time from 9.4 h to 29.6 h). Size-exclusion liquid chromatography and cytopathic effect inhibition assay indicated rhIFN preserved its structural and functional integrity in these chelates.