Laboratory Personnel

In order to build the proposed atlas, we used data from six post mortem cases from the body donor program of the University of Castilla - La Mancha (UCLM) Medical School (Albacete, Spain). Informed consent was obtained from the donors, following the Declaration of Helsinki. The use of this brain tissue for research purposes was approved by the Ethics Committee of the University Hospital of Albacete.
The demographic data of the cases is shown in Table 1. None of the donors had a history of disease that affected the morphology of the brain (or the thalamus): their clinical history did not include any confounding factors such substance abuse or pathology (e.g., dementia), and the weight of the specimens was approximately average. We acknowledge that the age range of these specimens is older than that of most neuroscience experiments. However, this issue is mitigated by the lack of pathology in the samples, as well as by the fact that our probabilistic atlas also includes an in vivo dataset of brain scans of 39 subjects (including several younger controls), as explained in Section Atlas Construction below. This strategy of combining these 39 in vivo scans with ex vivo images from elderly subjects has been proven successful by our earlier works on the hippocampus (Iglesias et al., 2015a (link)) and the amygdala (Saygin et al., 2017 (link)).Table 1

Demographics of the ex vivo cases that were used to build the atlas. PMI stands for “post mortem interval”.

Table 1

Case	Age at death	Gender	Brain weight	PMI
HNL4_13	97	male	1.238 Kg	9 h
HNL7_14	98	female	1.168 Kg	6 h
HNL5_13	59	male	1.020 Kg	N/A
HNL8_14	61	female	1.409 Kg	10 h
HNL14_15	87	male	1.100 Kg	2 h 30 m
HNL16_16	84	male	1.264 Kg	3 h 30 m

The fixation of all the brain samples was performed in situ by personnel of the UCLM Human Neuroanatomy Laboratory, by neck disection of both primitive carotids in the lower third of the neck, followed by cannulation of the carotids. The fixation started with a flush of 4 l of saline, followed by 8 l of 4% paraformaldehyde in phosphate buffer (pH 7.4). In order to allow the fixative to flow, the internal jugular vein was sectioned on one side. After perfusion, the brain was left in situ for 48 h, and subsequently extracted following standard autopsy procedures. Postfixation until scanning was carried out by storage in a container filled with 4% paraformaldehyde. This in situ fixation method better preserves the shape of the individual brain, fitting exactly the intracranial shape (as opposed to a generic container), and minimizes the impact of the extraction procedure on the probabilistic atlas to be built.

CaliciNet is a novel electronic laboratory surveillance network of local and state public health laboratories in the United States, coordinated by CDC. CaliciNet participants perform molecular typing of norovirus strains by using standardized laboratory protocols for reverse transcription PCR (RT-PCR) followed by DNA sequence analysis of the amplicons. A customized CaliciNet database developed in Bionumerics version 5.1 (Applied Maths, Austin, TX, USA) includes norovirus sequence and basic epidemiologic information (Table 1), which are submitted electronically via a secure connection to the CaliciNet server at CDC. Both epidemiologic and sequence data can then be used to help link multistate outbreaks to a common source (e.g., contaminated food). To ensure high-quality data entry, submissions to the CaliciNet server are performed by certified laboratory personnel of the participating state or local health laboratories, and final quality assurance/quality control is performed at CDC.
CaliciNet certification for participants is a 2-step process that involves evaluation of data entry and analysis of sequences and a laboratory panel test. Each laboratory must pass an annual proficiency test. The laboratory certification and proficiency test consists of analyzing a panel of fecal samples by real-time RT-PCR and conventional RT-PCR analysis followed by bidirectional sequencing as described below. Certified participants are then authorized to upload norovirus outbreak data consisting of >2 samples per outbreak to the national CaliciNet database (Table 1). GII.4 sequences with >2% and 3% difference in region C or D, respectively, and >10% difference with all other noroviruses are further analyzed at CDC by amplification of the VP1 or P2 region.

Participants were enrolled by randomization into two treatment groups according to the following steps. A master log of all screened participants was maintained. At screening, participants who signed the informed consent form and met all inclusion and none of the exclusion criteria were identified as eligible to be enrolled in the study. Eligible participants were assigned a sequential identification number when they presented to the clinic on Study Day 0. Statisticians at DF/Net Research (Seattle, WA) generated the list of randomized treatment assignments and included it in the enrollment module of the Interactive Web Randomization System (IWRS), which is integrated into the DFexplore software. The study coordinator or designee at the clinical site performed the enrollment and randomization using DFexplore. Block randomization of appropriate size was used to balance enrollment in a 1:1 ratio into each of the two groups. A designated individual at the study site was provided with a treatment key, which linked the treatment code to the actual treatment assignment and was kept in a secure place.
The study was conducted as a double-blind trial. Participants, investigators, study personnel performing any study-related assessments following study injection, and laboratory personnel performing immunology assays were blinded to treatment assignment. The randomization scheme from DF/Net Research was provided to unblinded study personnel (i.e., site pharmacists performing study product preparations and unblinded study product administrators).