Implementing a tertiary survey in the emergency general surgery population: Imitation is the sincerest form of flattery.

OBJECTIVES: The inherent complexity of the Emergency General Surgery (EGS) patient may preclude precise documentation at admission. To obviate lapses in documentation, an EGS tertiary survey (EGS-TS) was developed to enable early recognition of relevant omissions in documentation and clinical findings. We theorized that the creation of the EGS-TS would promote more thorough clinical documentation. METHODS: A prospective observational study was performed utilizing an EGS-TS from February 2019 through May 2019. The EGS-TS included physical exam, medication reconciliation, analysis of documentation for accuracy, and review of diagnostic imaging for incidental findings. RESULTS: There were 139 EGS admissions during the study period and 108 (78%) received an EGS-TS. Of those who received the EGS-TS, incorrect medication reconciliations (72%), incidental findings (12%), undocumented wounds (11%), and undocumented indwelling catheters were identified (6%). CONCLUSION: Implementation of an EGS-TS triggers a detailed evaluation and reveals opportunities for education, improved adherence to documentation standards, and further research that may guide quality improvement initiatives.

The impact of advanced practice providers on the surgical resident experience: Agree to disagree?

BACKGROUND: We examined and compared APP versus surgical resident perceptions of the role of APPs in surgical subspecialty teams. METHODS: Residents/first year surgical critical care fellows and inpatient service-specific APPs responded to a survey that examined perceptions about the APP-resident/fellow relationship. Statistical analysis compared responses using a Pearson chi-square test. RESULTS: Thirty-two resident/fellows (48%) and 10 APPs (42%) responded. There was consensus that having an APP on service decreases workload, contributes to continuity of care and enhances resident-patient coordination education and agreement that there was clear communication and adequate collaboration. Both groups differed with respect to APPs contribution to resident/fellow clinical education, role definition and chain of command. The majority of trainees felt that APPs function at a PGY2 level (51.7%) compared to APPs, who felt that they functioned at a PGY4/5 (22%) or Fellow (44%) level. CONCLUSION: APPs and resident/fellows agree that APPs impact resident workload, continuity of care and patient-coordination education.

Complete genome sequence of Nariva virus, a rodent paramyxovirus.

Nariva virus (NarPV) was isolated from forest rodents (Zygodontomys b. brevicauda) in eastern Trinidad in the early 1960s. Initial classification within the family Paramyxoviridae was based mainly on morphological observations including the structure of nucleocapsids and virion surface projections. Here, we report the characterization of the complete genome sequence of NarPV. The genome is 15,276 nucleotides in length, conforming to the rule-of-six, and has a genome organization typical of most members of the family, with six transcriptional units in the order 3'-N-P-M-F-H-L-5'. The gene junctions contain highly conserved gene start and stop signals and a tri-nucleotide intergenic sequence present in most members of the subfamily Paramyxovirinae. Sequence comparison studies indicate that NarPV is most closely related to Mossman virus, which was isolated from wild rats (Rattus leucopus) in Queensland, Australia, in 1970. This study confirmed the classification of NarPV as a member of the subfamily Paramyxovirinae and established the close genome organization and sequence relationship between the two rodent paramyxoviruses isolated almost a decade apart and from two locations separated by more than 15,000 km.

Viral morphogenesis and morphological changes in human neuronal cells following Tioman and Menangle virus infection.

Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10(1)) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.

A randomised controlled trial examining the longer-term outcomes of standard versus new antiepileptic drugs. The SANAD trial.

OBJECTIVES: To compare clinicians' choice of one of the standard epilepsy drug treatments (carbamazepine or valproate) versus appropriate comparator new drugs. DESIGN: A clinical trial comprising two arms, one comparing new drugs in carbamazepine and the other with valproate. SETTING: A multicentre study recruiting patients with epilepsy from hospital outpatient clinics. PARTICIPANTS: Patients with an adequately documented history of two or more clinically definite unprovoked epileptic seizures within the last year for whom treatment with a single antiepileptic drug represented the best therapeutic option. INTERVENTIONS: Arm A was carbamazepine (CBZ) versus gabapentin (GBP) versus lamotrigine (LTG) versus oxcarbazepine (OXC) versus topiramate (TPM). Arm B valproate (VPS) versus LTG versus TPM. MAIN OUTCOME MEASURES: Time to treatment failure (withdrawal of the randomised drug for reasons of unacceptable adverse events or inadequate seizure control or a combination of the two) and time to achieve a 12-month remission of seizures. Time from randomisation to first seizure, 24-month remission of seizures, incidence of clinically important adverse events, quality of life (QoL) outcomes and health economic outcomes were also considered. RESULTS: Arm A recruited 1721 patients (88% with symptomatic or cryptogenic partial epilepsy and 10% with unclassified epilepsy). Arm B recruited 716 patients (63% with idiopathic generalised epilepsy and 25% with unclassified epilepsy). In Arm A LTG had the lowest incidence of treatment failure and was statistically superior to all drugs for this outcome with the exception of OXC. Some 12% and 8% fewer patients experienced treatment failure on LTG than CBZ, the standard drug, at 1 and 2 years after randomisation, respectively. The superiority of LTG over CBZ was due to its better tolerability but there is satisfactory evidence indicating that LTG is not clinically inferior to CBZ for measures of its efficacy. No consistent differences in QoL outcomes were found between treatment groups. Health economic analysis supported LTG being preferred to CBZ for both cost per seizure avoided and cost per quality-adjusted life-year gained. In Arm B for time to treatment failure, VPS, the standard drug, was preferred to both TPM and LTG, as it was the drug least likely to be associated with treatment failure for inadequate seizure control and was the preferred drug for time to achieving a 12-month remission. QoL assessments did not show any between-treatment differences. The health economic assessment supported the conclusion that VPS should remain the drug of first choice for idiopathic generalised or unclassified epilepsy, although there is a suggestion that TPM is a cost-effective alternative to VPS. CONCLUSIONS: The evidence suggests that LTG may be a clinical and cost-effective alternative to the existing standard drug treatment, CBZ, for patients diagnosed as having partial seizures. For patients with idiopathic generalised epilepsy or difficult to classify epilepsy, VPS remains the clinically most effective drug, although TPM may be a cost-effective alternative for some patients. Three new antiepileptic drugs have recently been licensed in the UK for the treatment of epilepsy (levetiracetam, zonisamide and pregabalin), therefore these drugs should be compared in a similarly designed trial.

Bats, civets and the emergence of SARS.

Severe acute respiratory syndrome (SARS) was the first pandemic transmissible disease of previously unknown aetiology in the twenty-first century. Early epidemiologic investigations suggested an animal origin for SARS-CoV. Virological and serological studies indicated that masked palm civets ( Paguma larvata), together with two other wildlife animals, sampled from a live animal market were infected with SARS-CoV or a closely related virus. Recently, horseshoe bats in the genus Rhinolophus have been identified as natural reservoir of SARS-like coronaviruses. Here, we review studies by different groups demonstrating that SARS-CoV succeeded in spillover from a wildlife reservoir (probably bats) to human population via an intermediate host(s) and that rapid virus evolution played a key role in the adaptation of SARS-CoVs in at least two nonreservoir species within a short period.

Full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the Americas.

Mapuera virus (MPRV) was isolated from a fruit bat in Brazil in 1979, but its host range and disease-causing potential are unknown. Porcine rubulavirus (PoRV) was identified as the aetiological agent of disease outbreaks in pigs in Mexico during early 1980s, but the origin of PoRV remains elusive. In this study, the completed genome sequence of MPRV was determined, and the complete genome sequence of PoRV was assembled from previously published protein-coding genes and the non-coding genome regions determined from this study. Comparison of sequence and genome organization indicated that PoRV is more closely related to MPRV than to any other members of the genus Rubulavirus. In the P gene coding region of both viruses, there is an ORF located at the 5' end of the P gene overlapping with the P protein coding region, similar to the C protein ORF present in most viruses of the subfamily Paramyxovirinae, but absent in other known rubulaviruses. Based on these findings, we hypothesise that PoRV may also originate from bats, and spillover events from bats to pigs, either directly or via an intermediate host, were responsible for the sporadic disease outbreaks observed in Mexico.

Prion-removal capacity of chromatographic and ethanol precipitation steps used in the production of albumin and immunoglobulins.

BACKGROUND AND OBJECTIVES: Although there is no epidemiological evidence to suggest that classical Creutzfeldt-Jakob disease (CJD) is transmitted through blood or blood products, the variant form (vCJD) has been implicated in transmission via packed red blood cells. The potential threat of the infectious agent contaminating plasma pools has led to manufacturing processes being examined for capacity to remove prions. The objective of these studies was to examine the prion-removal potential of the chromatographic purification and ethanol precipitation steps used to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: Western blot assay was used to examine the partitioning of proteinase K-resistant scrapie prion protein (PrPsc) over DEAE Sepharose, CM Sepharose and Macro-Prep High Q chromatographic columns, utilizing microsomal scrapie 263K spiked into each scaled down feedstream and assayed after each chromatographic step. In further studies, bioassay in C57 black mice was used and spikes of 10 000 g clarified brain homogenate of scrapie ME7 were added to feedstreams before sequences of scaled down chromatographic or Cohn fractionation process steps. RESULTS: The microsomal spiking study with Western blot detection demonstrated substantial partitioning of PrPsc away from the target proteins in all ion exchange chromatographic steps examined. The log10 reduction factors (LRF) across DEAE Sepharose and CM Sepharose columns for albumin were > or = 4.0 and > or = 3.0 respectively. The reductions across DEAE Sepharose and Macro-Prep High Q for intravenous immunoglobulin were 3.3 and > or = 4.1 respectively. Bioassay demonstrated LRFs of >or = 5.6 across the combination of DEAE Sepharose and CM Sepharose columns in the albumin process and > or = 5.4 across the combination of DEAE Sepharose and Macro-Prep High Q columns in the intravenous immunoglobulin process. Bioassay studies also demonstrated a LRF of > or = 5.6 for immunoglobulin produced by Cohn fractionation. CONCLUSIONS: Using rodent-adapted scrapie as a model, the studies indicated that ion exchange chromatography, as well as Cohn immunoglobulin fractionation have the potential to effectively reduce the load of TSE agents should they be present in plasma pools.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Analysis of speech-related variance in rapid event-related fMRI using a time-aware acquisition system.

Speech production introduces signal changes in fMRI data that can mimic or mask the task-induced BOLD response. Rapid event-related designs with variable ISIs address these concerns by minimizing the correlation of task and speech-related signal changes without sacrificing efficiency; however, the increase in residual variance due to speech still decreases statistical power and must be explicitly addressed primarily through post-processing techniques. We investigated the timing, magnitude, and location of speech-related variance in an overt picture naming fMRI study with a rapid event-related design, using a data acquisition system that time-stamped image acquisitions, speech, and a pneumatic belt signal on the same clock. Using a spectral subtraction algorithm to remove scanner gradient noise from recorded speech, we related the timing of speech, stimulus presentation, chest wall movement, and image acquisition. We explored the relationship of an extended speech event time course and respiration on signal variance by performing a series of voxelwise regression analyses. Our results demonstrate that these effects are spatially heterogeneous, but their anatomic locations converge across subjects. Affected locations included basal areas (orbitofrontal, mesial temporal, brainstem), areas adjacent to CSF spaces, and lateral frontal areas. If left unmodeled, speech-related variance can result in regional detection bias that affects some areas critically implicated in language function. The results establish the feasibility of detecting and mitigating speech-related variance in rapid event-related fMRI experiments with single word utterances. They further demonstrate the utility of precise timing information about speech and respiration for this purpose.

Pulau virus; a new member of the Nelson Bay orthoreovirus species isolated from fruit bats in Malaysia.

After the outbreak of Nipah virus (NiV) in 1998-99, which resulted in 105 human deaths and the culling of more than one million pigs, a search was initiated for the natural host reservoir of NiV on Tioman Island off the east coast of Malaysia. Three different syncytia-forming viruses were isolated from fruit bats on the island. They were Nipah virus, Tioman virus (a novel paramyxovirus related to Menangle virus), and a reovirus, named Pulau virus (PuV), which is the subject of this study. PuV displayed the typical ultra structural morphology of a reovirus and was neutralised by serum against Nelson Bay reovirus (NBV), a reovirus isolated from a fruit bat (Pteropus poliocephalus) in Australia over 30 years ago. PuV was fusogenic and formed large syncytia in Vero cells. Comparison of dsRNA segments between PuV and NBV showed distinct mobility differences for the S1 and S2 segments. Complete sequence analysis of all four S segments revealed a close relationship between PuV and NBV, with nucleotide sequence identity varying from 88% for S3 segment to 56% for the S1 segment. Similarly phylogenetic analysis of deduced protein sequences confirmed that PuV is closely related to NBV. In this paper we discuss the similarities and differences between PuV and NBV which support the classification of PuV as a novel mammalian, fusogenic reovirus within the Nelson Bay orthoreovirus species, in the genus Orthoreovirus, family Reoviridae.

RNA synthesis during infection by Hendra virus: an examination by quantitative real-time PCR of RNA accumulation, the effect of ribavirin and the attenuation of transcription.

Hendra virus is one of two virus species within the newly-formed genus Henipavirus, subfamily Paramxyovirinae. It is a designated select agent with potential biosecurity threat to both human and animal health. Quantitative real-time PCR was used to measure viral RNA synthesis in Vero cells infected by Hendra virus, and to examine the inhibitory effect of ribavirin. It was also used to determine the points of attenuation during transcription of the six viral genes N, P, M, F, G and L by targeting amplicons located towards the 3' end of each gene. Major increases in viral RNA and virus yield occurred between 4 to 8 h and 8 to 10 h post infection, respectively. The effect of ribavirin was examined at a range of concentrations up to 400 microm. At 50 microm, RNA synthesis was reduced 9 fold, and virus yield 58 fold. As expected for a member of the order Mononegavirales, a gradient of transcription was observed in Hendra virus-infected cells. There was significant attenuation at the M-F and G-L junctions, more closely resembling Sendai virus (genus Respirovirus) than measles virus (genus Morbillivirus).

Henipaviruses: recent observations on regulation of transcription and the nature of the cell receptor.

Hendra virus (HENV) and Nipah virus (NIPV) are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genetic and biological characteristics that differentiate henipaviruses from other members of the subfamily are summarized. Although they do not display neuraminidase and hemagglutination activities and in that regard resemble viruses in the genus Morbillivirus, several recent observations highlight similarities between henipaviruses and respiroviruses (genus Respirovirus) in structure and replication strategy. First, three-dimensional modeling studies suggest that the external globular head domain of the HENV G protein resembles that of respiroviruses rather than morbilliviruses. Second, the pattern of transcriptional attenuation in HENV-infected cells resembles that observed with Sendai virus, a respirovirus, and differs from that found in cells infected with measles virus, a morbillivirus. Henipaviruses have a broad host range in vitro and in vivo, indicating wide distribution of cellular receptor molecules. The extensive host range has been confirmed in a quantitative in vitro cell-fusion assay using recombinant vaccinia viruses expressing the attachment and fusion proteins of HENV and NIPV. Cell lines of diverse origin and which are permissive in the in vitro cell fusion assay have been identified and the pattern of relative susceptibilities is the same for both HENV and NIPV, implying that both viruses use the same cell receptor. Protease treatment of permissive cells destroys their ability to fuse with cells expressing viral envelope glycoproteins. Virus overlay protein binding assay (VOPBA) and radio-immune precipitation assays confirm that both HENV and NIPV bind to membrane proteins in the 35-50 kD range. Treatment of cell membrane proteins with N-glycosidase eliminates HeV binding activity in VOPBA whereas treatment with neuraminidase has no effect on binding. Thus preliminary evidence suggests that NIPV and HENV bind to the same glycoprotein receptor via a non-sialic acid-dependant mechanism.

Genetic diversity of bluetongue viruses in south east Asia.

Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.

Regional overview of bluetongue viruses in South-East Asia: viruses, vectors and surveillance.

Structured epidemiological studies based on sentinel herds in Indonesia and Malaysia have provided much information regarding the bluetongue (BT) viruses (BTV) and their likely vectors in South-East Asia. Serotypes 1, 2, 3, 7, 9, 12, 16, 21 and 23 have been isolated. Molecular analyses show all group within the Australasian topotype, with four genotypic sub-groupings identified to date. There are relationships to isolates from both India and Australia. Strains of BTV in South-East Asia do not appear to be highly virulent, since BT disease is not seen in local sheep. Known vector species identified include Culicoides fulvus, C. actoni, C. wadai and C. brevitarsis. C. imicola has not been identified in Malaysian or Indonesian studies. Molecular analyses indicate movement of South-East Asian strains of BTV into northern Australia, and the gradation in observations between India and eastern Australia regarding serotype, genotype, virulence and vector species suggests movement along a conceptual gradient through South-East Asia.

Genetic diversity of bluetongue viruses in Australasia.

The authors have characterised the genetic diversity of the bluetongue virus (BTV) RNA segments 3 and 10 from Indonesia, Malaysia and Australia. Analysis of RNA segment 3, which codes for the core protein VP3, showed conserved sequences in the previously defined Australasian topotype, but which further divided into four distinct clades or genotypes. Certain genotypes appeared to be geographically restricted while others were distributed widely throughout South-East Asia. Ongoing surveillance programmes in Australia have identified the movement of Indonesian genotypes into northern Australia and possible reassortment among them. Similarly, analysis of RNA segment 10, which codes for the non-structural protein NS3/3A, showed they were also conserved and grouped into five clades or genotypes, three Asian and two North American/South African.

Characterisation and monitoring of neutralisation-resistant VP2 phenotypes in BTV-1 isolates from northern Australia collected over a twenty-year period.

Phenotypic profiles of the VP2 protein of isolates of bluetongue virus serotype 1 (BTV-1) collected from Queensland and the Northern Territory, Australia, between 1979 and 1986 were analysed using neutralising monoclonal antibodies (MAbs) raised to the prototype isolate of Australian BTV-1 collected in the Northern Territory in 1979. Two distinct profiles were found. Northern Territory isolates exhibited the prototype profile, yet those from Queensland had a significantly different ('resistant') profile. Nucleotide sequencing of gene segment 2 from both groups of isolates was undertaken. When the nucleotide sequences of isolates from a later period in each State were analysed (1997-2001), all exhibited the 'resistant' profile. Thus, a novel VP2 phenotype, already in existence in Queensland, had supplanted a pre-existing VP2 phenotype in the Northern Territory between the two periods. Furthermore, amino acid differences between the resistant and prototype VP2 proteins were analogous to amino acid substitutions known to be associated with neutralisation resistance. The host immune response may therefore have contributed to selection of the 'resistant' phenotype.

Developing new orbivirus diagnostic platforms.

Traditionally, successful orbivirus identification and characterisation has been dependent upon the development and application of techniques for virus isolation. In recent years however polymerase chain reaction (PCR)-based molecular detection systems have revolutionised medical infectious disease diagnosis and in some instances have removed the requirement to isolate pathogens for confirmation of clinical diagnoses. In multiplexing formats, PCR-based methods also have the capacity to detect novel pathogens and variants of existing pathogens. Detection and characterisation of veterinary pathogens such as bluetongue virus will follow the same evolutionary path. Work is underway in a number of laboratories to develop the infrastructure and databases required to permit the use of DNA-based molecular systems for orbivirus detection and characterisation. Novel multiplexed protein analysis platforms also offer opportunities to not only enhance the speed and sensitivity of serological assays but also permit the development of serological procedures that a few years ago were not technically feasible.

Full length genome sequence of Tioman virus, a novel paramyxovirus in the genus Rubulavirus isolated from fruit bats in Malaysia.

A novel paramyxovirus in the genus Rubulavirus, named Tioman virus (TiV), was isolated in 1999 from a number of pooled urine samples of Island Flying Foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. TiV is antigenically related to Menangle virus (MenV) that was isolated in Australia in 1997 during disease outbreak in pigs. Sequence analysis of the full length genome indicated that TiV is a novel member of the genus Rubulavirus within the subfamily Paramyxovirinae, family Paramyxoviridae. However, there are several features of TiV which make it unique among known paramyxoviruses and rubulaviruses in particular: (1) TiV, like MenV, uses the nucleotide G as a transcriptional initiation site, rather than the A residue used by all other known paramyxoviruses; (2) TiV uses C as the +1 residue for all intergenic regions, a feature not seen for rubulaviruses but common for all other members within the subfamily Paramyxovirinae; (3) Although the attachment protein of TiV has structural features that are conserved in other rubulaviruses, it manifests no overall sequence homology with members of the genus, lacks the sialic acid-binding motif N-R-K-S-C-S and has only two out of the six highly conserved residues known to be important for the catalytic activity of neuraminidase.

Biosynthesis and secretion of pituitary hormones: dynamics and regulation.

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to